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Assessment of soil potential to natural attenuation and autochthonous bioaugmentation using microarray and functional predictions from metagenome profiling

Assessment of soil potential to natural attenuation and autochthonous bioaugmentation using... Purpose The use of autochthonous microorganisms for the bioaugmentation of areas contaminated with hydrocarbons has a high potential to overcome the limitations associated with the difficulty of allochthonous microorganisms to adapt. The prediction of bioremediation effects of autochthonous bioaugmentation can be improved by employing the rapid methods of the direct detection of genes crucial to the hydrocarbon biodegradation. This study aimed to evaluate the potential of microflora originating from soils with different levels of anthropogenization for application in autochthonous bioaugmentation by using microarray and functional predictions from metagenome profiling. Methods Analyses based on the modern techniques of molecular biology—DNA microarrays and next-generation sequencing— coupled with the functional predictions of metagenome profiling. Results Studies indicated that the metapopulations of all analyzed stations possess the ability to biodegrade petroleum hydrocarbons. It was established that the long-term supply of hydrocarbons in the areas characterized by strong anthropogenization resulted in increasing the biological decomposition of aromatic and polycyclic aromatic com- pounds. In contrast, areas with a low level of anthropogenization were characterized by a higher potential to decompose aliphatic hydrocarbons. Although alpha-biodiversity decreased when the consortia was isolated and cul- tivated under laboratory conditions with hydrocarbons as the sole carbon source, microbial communities with genetic biodegradation potential increased, which was confirmed by the analysis involving the loss of selected hydrocarbon fractions in aqueous systems. Conclusions The presented studies indicated the vast potential for the application of isolated autochthonous microflora on soils permanently contaminated with hydrocarbons. The prediction of bioremediation effects may be improved by employing the rapid method of the direct detection of genes crucial to the biological decomposition of hydrocarbons, with DNA microarrays developed in the framework of this study. . . . Keywords Autochthonous bioaugmentation Microarray Hydrocarbon biodegradation Biodiversity Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-019-01486-3) contains supplementary material, which is available to authorized users. * Jakub Czarny Department of Biochemistry and Biotechnology, Poznan University pubjc@igs.org.pl of Life Sciences, Dojazd 11, 60-632 Poznan, Poland Department of Rare Earths, Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznan, Poland Institute of Forensic Genetics, Al. Mickiewicza 3/4, 85-071 Bydgoszcz, Poland Department Biotechnology and Food Microbiology, Poznan University of Life Sciences, Wojska Polskiego 48, Institute of Food Technology of Plant Origin, Poznan University of 60-627 Poznan, Poland Life Sciences, Wojska Polskiego 31, 60-624 Poznan, Poland 946 Ann Microbiol (2019) 69:945–955 Introduction molecular metagenomics tools that will allow for their assess- ment (Sierra-Garcia et al. 2014). Increased demand for solid fossil fuels, their intense explora- This study aimed to evaluate the potential of microflora orig- tion and distribution result in the unintended release of petro- inating from soils with different levels of anthropogenization and leum hydrocarbons to the natural environment (Adams et al. supply of petroleum hydrocarbons for the biodegradation of se- 2015; Singh et al. 2017; Zivelytea et al. 2017). It is believed lected fractions of hydrocarbons based on the modern techniques that hydrocarbons are among the most widespread contami- of molecular biology—namely DNA microarrays and next- nants in highly industrialized and developing countries generation sequencing coupled with the functional prediction of (Macaulay and Rees 2014). Local contamination of soil may metagenome profiling. Additionally, the ability of consortia to result from accidental leaks during the extraction, refining, proliferate under laboratory conditions and the biodegradation transport, and storage of fossil fuels, as well as during the efficiency of hydrocarbons under model conditions was ana- improper storage of petroleum products in underground tanks, lyzed. This analysis allowed the identification of the diverse en- the destruction of industrial pipelines and the illegal uptake of zymatic capabilities of soil systems and the determination of the fuels (Das and Chandran 2011). Biological methods based on possibility of using the ABA technology in cases of widespread the natural ability of microorganisms to decompose organic contaminations. compounds enzymatically comprise a promising remediation technology for contaminated areas. These methods are an al- ternative for chemical and physical methods and are charac- Methods terized by their low operational costs and limited risk of trans- formation of xenobiotics to more toxic intermediates (Karigar Soil sampling and Rao 2011; Kumar et al. 2011; Macaulay and Rees 2014). Bioaugmentation is a strategy for the biological decomposi- Three main types of study areas were defined: post-industry tion of petroleum hydrocarbons, and this strategy has been areas with a high level of anthropogenization (A), moderate known since the 1970s and is based on the introduction of level of anthropogenization (B), and low level of microbial populations characterized by a high biodegradation anthropogenization under the statutory form of natural protec- potential. This method is particularly promising in cases in tion (C). In each area, a sampling site was established which microorganisms do not possess the proper metabolic (supplementary materials, Table S1)withan area of 5 m× predispositions to biologically decompose petroleum hydro- 5 m. A composite sample was gathered from each site (com- carbons or their ratio in the population is relatively low posed of 10 partial samples of topsoil layer) using a Shelby (Adams et al. 2015). The efficiency of introducing allochtho- tube sampler (2-cm diameter, 10-cm depth). Additionally, a nous microorganisms with high biodegradation potential is model system for soil contamination with a low level of broadly discussed in the literature. It is believed that the lim- anthropogenization was prepared. Approx. 50 g was taken ited ability of the inoculated consortium to adapt to local con- from the composite soil sample, and then, 5 g of diesel oil ditions and the rivalry for the environmental niche with au- (DO) was added and left at room temperature for 2 months. tochthonous microflora are the factors that are responsible for The soils with the addition of DO were labeled type D the long-term reduction in biodegradation efficiency (Ueno (supplementary materials, Table S1). The selected hydrocar- et al. 2007; Macaulay and Rees 2014). Several authors indi- bon concentrations of the soil samples were presented as a cate that the effect of the stimulation of biodegradation effi- supplementary material, Table S2. ciency does not occur or is only short-term and that it is insig- nificant in the removal of hydrocarbons (Bento et al. 2005; Isolation of consortia and preparation Szczepaniak et al. 2016). Furthermore, it is believed that the of the inoculum efficiency of biodegradation processes is correlated, not with biodiversity, but rather with the number of individual micro- Approx. 10 g of soil was taken from each sample, added to organisms, which are capable of decomposing petroleum hy- 90 mL of 0.9% solution of NaCl and shaken for 30 min drocarbons (Wu et al. 2016). A novel method to overcome the (150 rpm). Next, the systems were left for 10 min to allow limitations associated with the adaptation of allochthonous for the sedimentation of mineral particles, and then, 1 mL of microorganisms may be the autochthonous bioaugmentation the water phase was transferred to flasks containing tryptic soy (ABA) technique, which is based on the re-introduction of broth (common nutritional medium to supports the growth of local microflora. To date, there are only a few research studies a wide range of bacteria) (Wanger et al. 2017) with the addi- involving this field (Ueno et al. 2007; Nikolopoulou et al. tion of 2% of glucose. The obtained consortia (ConA-ConD) 2013). Since the efficiency of all bioremediation techniques, were grown for 72 h under aerobic conditions on a shaker including ABA, is correlated with the enzymatic potential of (130 rpm), then centrifuged (10 min, 4000 rpm), and rinsed autochthonous microflora, there is a need to develop three times with 0.9% solution of NaCl, centrifuging each Ann Microbiol (2019) 69:945–955 947 time according to the described procedure. The final inoculum follows: interface temperature 230 °C, scanning interval 0.15 s, was suspended in 0.9% solution of NaCl by normalizing the and scanning range 33–250 m/z. concentration based on optical density OD (600) to the value of 0.6 (Helios Delta Vis, ThermoFisher Scientific Inc., USA). Assessment of viability of consortia Biodegradation of hydrocarbons in model systems Samples were taken from the model system after 24, 48, 72, Biodegradation was conducted in Duran-type flasks with baf- and 168 h of biodegradation. To determine the viability of fles, in model aqueous systems with the addition of diesel oil consortia, a commercially available ADP/ATP Ratio Assay as the sole carbon source. The experimental system was com- Kit (Sigma-Aldrich Inc.) was used. Approx. 10 μLof each posed of 50 mL of mineral medium with microelements sample was transferred to a 96-well plate, and then, the ATP (Na HPO 6.21 g/L, KH PO 2.8 g/L, NaCl 0.5 g/L, NH Cl reagent (prepared in accordance with the manufacturers’ pro- 2 4 2 4 4 1.0 g/L, MgSO ×7H O 0.01 g/L, FeSO ×7H O 0.001 g/L, tocol) was added. The samples were incubated for 60 s at 4 2 4 2 MnSO ×4H O 0.0005 g/L, ZnCl 0.00064 g/L, CaCl × room temperature, and then, the luminescence (RLUa) was 4 2 2 2 6H O 0.0001 g/L, BaCl 0.00006 g/L, CoSO ×7H O measured using a SpectraMax M2e multi-mode plate reader 2 2 4 2 0.000036 g/L, CuSO ×5H O 0.000036 g/L, H BO (Molecular Devices Inc.). The samples were incubated for 4 2 3 3 0.00065 g/L, H MoO 0.005 g/L, EDTA 0.001 g/L, HCl another 10 min, and the luminescence (RLUb) was measured 2 4 37% 0.0146 mL/L), 1 g of diesel oil, and 250 μLofthe again. Next, 5 μL of the ADP Reagent (prepared in accor- microbial inoculum. Biodegradation was conducted for 7 days dance with the manufacturers’ protocol) was added. After at 25 °C under aerobic conditions on a shaker (130 rpm). Each 60 s, the luminescence (RLUc) was measured for the third sample was prepared in six repetitions. Three were used for time. The ADP/ATP ratio was calculated based on the follow- the further analysis of hydrocarbon biodegradation, and three ing formula: were used for the analysis of the viability of consortia. Additionally, three internal controls without the addition of ADP RLUc−RLUb ratio ¼ microorganisms were prepared to exclude the abiotic loss of ATP RLUa hydrocarbons. Analysis of the biodegradation of selected fractions Isolation of DNA and sequencing of hydrocarbons Isolation of DNA After 7 days of the biodegradation process, the samples were subjected to extraction. Approx. 2.5 mL of acetone The isolation of the genetic material from the soil samples and was added to each system and shaken for 10 min the liquid cultures was conducted using the following kits: (150 rpm). Next, 200 μL of internal standards was intro- Genomic Mini AX Soil Spin (A&A biotechnology Inc.) and duced: n-nonane (Sigma-Aldrich Inc.), n-octacosane Genomic Mini AX Bacteria Spin (A&A Biotechnology Inc.), (Restek Inc.), and acenaphthene-d10 (Restek Inc.) with respectively, by following the manufacturers’ recommendations. concentrations of 6 mg/mL, 6 mg/mL, and 1.5 mg/mL, The validation of isolation efficiency was carried out with a respectively, in an acetone/hexane mixture (2:3 v/v). fluorometric method using a Qubit 3.0 apparatus and Qubit™ Then, 25 mL of hexane (Poch Inc.) was added, and the dsDNA HS Assay Kit (ThermoFisher Scientific Inc.). samples were shaken again for 60 min (150 rpm). The samples were stabilized for 30 min until the phases were separated, and 2 mL of acetone (Poch Inc.) was added PCR amplification and sequencing dropwise to remove any emulsions. The prepared extracts were diluted 25-fold in hexane and subjected to GC-MS Universal prokaryotic primers 515F-806R were used to am- analysis. plify the V4 region of 16S rRNA (Caporaso et al. 2012). The The GC-MS analysis was carried out using a Shimadzu 17A PCR reaction was carried out in a volume of 25 μL(5 μL gas chromatograph coupled with a QP5000 mass spectrometer microbial template genomic DNA, 5 μL of each primer, equipped with a Rxi-5MS COLUMN (Restek Inc.). The follow- 2.5 μL of PCR-grade water (ThermoFisher Scientific Inc.), ing separation conditions were employed: carrier gas (helium) and 12.5 μL of PCR Master Mix with the Taq polymerase flow 1.1 mL/min, injection port temperature 250 °C, splitless (ThermoFisher Scientific Inc.). The following PCR reaction injection mode, injection volume 1.0 μL, and column tempera- conditions were employed: initial denaturation 95 °C for ture program 40 °C held for 1 min, ramped at rate of 15 °C/min 3 min; 35 cycles 1 min at 95 °C, 30 s at 52 °C, and 1 min at to 300 °C, and held for 7 min. Detection conditions were as 72 °C; and final extension at 72 °C for 10 min. The amplicons 948 Ann Microbiol (2019) 69:945–955 were purified using Clean-Up columns (A&A Biotechnology the following sequence: 5′-C12-AMINO-(dT)18-ACG TAC Inc.) and then used for the construction of libraries. GTA CGT ACG TAC GTA CGT-Cy5-3′. Sequencing was conducted using a MiSeq (Illumina Inc., All four sub-matrices are printed on the glass using the CA) apparatus with a MiSeq Reagent Kit v2 (2 × 250 bp) same system. Upon application of the probes, the binding (Illumina Inc.). Details regarding the preparation of libraries process was conducted for 12 h at room temperature and hu- were described in a previous study (Szczepaniak et al. 2016). midity < 30%, and then, the systems are rinsed two times with a 0.1% solution of sodium dodecyl sulfate and two more times Analysis of data after sequencing with water. Raw data in the FASTQ format were imported to the CLC DNA extraction and amplification Genomics Workbench 8.5 software with the CLC Microbial Genomics Module 1.2 (Qiagen Inc.). The reads were DNA was isolated from the soil and batch cultures as de- demultiplexed, and paired ends were merged (mismatch scribed in the previous section. cost = 2, min score = 8, Gap cost = 3, max unaligned end mis- PCR amplification was conducted in two multiplex reac- matches = 5). Next, the primer sequences were trimmed (qual- tions. Two different fragments were amplified for each gene, ity limit = 0.05, ambiguous limit = N), and the identification and one was amplified for each multiplex reaction; they were and elimination of the chimeric reads were conducted. The detected using complementary probes (supplementary output data were clustered independently based on two refer- materials, Table S3). Multiplex 1 and 2 PCR amplifications ence databases: SILVA v119 (Quast et al. 2013)and were conducted in a 50 μL volume using specific starters GreenGenes 13.5 (DeSantis et al. 2006) at a 97% probability (supplementary materials, Table S3) at the following condi- level of open taxonomic units (OTUs). Based on the merged tions: 1× PCR buffer, MgCl 2.5 mM, dNTPs 250 μM(A&A abundance table (clustered against SILVA v119), the alpha- Biotechnology Inc.), BSA FractionV 0.16 mg/mL (Sigma- biodiversity (number of OTUs) and beta-biodiversity (Bray- Aldrich Inc.), Glycerol (10% w/v), 5 U TaqPol (A&A Curtis PCoA) factors were determined. Biotechnology Inc.), starters based on the Table S3 (supple- To predict the gene content from each OTUs table con- mentary materials), and 10 μL of DNA. The following tem- structed against GreenGenes 13.5, the Phylogenetic perature profile was used for the reaction: initial denaturation Investigation of Communities by Reconstruction of at 95 °C for 3 min, and then, 35 cycles denaturation at 94 °C Unobserved States (PICRUSt) tool was used (Langille et al. for 1 min, annealing 60 °C for 1 min, elongation 72 °C for 2013). The output data were a collection involving the abun- 1 min, and final elongation 60 °C for 15 min. Two studied dance of key functional orthologues (KO) in each sample. samples are subjected to amplification—namely negative con- Based on the Kyoto Encyclopedia of Genes and Genomes trol for DNA isolation and positive control for PCR (KEGG), the KO in hydrocarbon biodegradation was selected, amplification. which allowed the prediction of the functional pathways The amplification products of Multiplex 1 and 2 were (Kanehisa and Goto 2000). Finally, the data were grouped pooled and purified from the starters and nucleotides using based on the EC classification. To compare the samples, the the precipitation method on the clean-up columns (A&A results were normalized by dividing the total predicted gene Biotechnology Inc.). The purified products were rinsed from abundance for the number of OTUs in each sample. the column using a volume of 50 μL. Each test study includes the analysis of the positive control, the negative control, and Microarrays the studied sample (two replications). Array preparation Matrix analysis Four round areas with a diameter of 9 mm (sub-matrices) were A60-μL aliquot of the reaction mixture was applied to the separated on SUPEROXY glass using 0.25-mm foil. separated submatrices for the positive control, the negative Oligonucleotides with a sequence complementary to the am- control, and the studied samples. The reaction mixture plified sequences were applied to fixed places on the four contained the following: 1× SBE Buffer, 0.75 μMof 7- areas by the contact method using SpotBot 3 Microarrayer propargylamino-7-deaza-2′,3′-dideoxyadenosine-5′ (Arrayit Inc.). Probe sequences were established using thiphosphate-6-FAM, 5-propargylamino-2 ′,3 ′- Primer3 Plus software (supplementary materials, Table S3) dideoxycytidine-5′thiphosphate-6-FAM, 7-propargylamino- (Untergasser et al. 2007). On the 5′ end, each probe would 7-deaza-2′3′dideoxyguanosine-5′triphosphate6-FAM, 5- possess a C12-AMINO modification and 18dT sequence pre- propargylamino-2′,3′-dideoxyuridine-5′triphosphate-6-FAM, ceding the correct oligonucleotide sequence. In each 5-propargylamino-2′,3′-dideoxyuridine-5′triphosphate-6- submatrix, a control for matrix preparation is applied with FAM (Jena Bioscience Inc.), 5 U DynaSeq DNA Polymerase Ann Microbiol (2019) 69:945–955 949 (Finnzyme Inc.), and 20 μL of the purified PCR product. The hydrocarbons (TPH) into account, the highest efficiency of the matrix was covered with foil for coating PCR plates. biological decomposition of diesel oil was observed in the Amplification was conducted in a thermocycler equipped with case of ConC. a microscopic glass amplification unit (Dual Flat Block GeneAmp PCR System 9700 Life Technologies Inc., Assessment of viability of consortia Mastercycler Nexus Flat Eppendorf Inc.) at the following con- ditions: 30 cycles denaturation at 94 °C for 1 min, annealing/ To estimate the viability and proliferation of consortia, changes in elongation 50 °C for 1 min, and over temperature 50 °C. the ADP/ATP ratio (supplementary materials, Fig. S1) were de- After the termination of the reaction, the matrix was termined. All consortia after 24 h of biodegradation entered a rinsed using a High Throughput Wash Station (Arrayit phase of exponential growth. The lowest ADP/ATP ratio was Inc.) twice for 2 min in deionized water and dried by noted in the case of ConB. centrifugation for 1 min at 500g in a Microarray High- Speed Centrifuge (Arrayit Inc.). Metapopulation analysis The matrices were analyzed using a GenePix 4300A scan- ner (Molecular Devices Inc.) in an ozone-free cell at excitation Taxonomic identification using the SILVA v119 database and emission wavelengths of 492 and 517 nm, respectively, based on the V4 region of 16S rRNA allowed for a summary for 6-FAM and 625 and 670 nm, respectively, for Cy-5 (spot- detection of 5 phyla, 10 classes, 23 orders, 61 families, 152 ting control). genera, and 389 species of microorganisms. The structure of the metapopulations of the soils and consortia cultivated under Statistical analysis laboratory conditions is presented in Fig. 2. Proteobacteria was the dominant phylum in all samples. The presence of To evaluate the significance of the differences among the an- Cyanobacteria was observed only in SoilB (0.47%). Taking alyzed systems, a nonparametric Kruskal–Wallis test was the comparison of the taxonomic composition of soils and the employed. Mann-Whitney test was used to test differences corresponding consortia into account, the highest differences between groups. All data represent the mean and the standard were observed in the case of Proteobacteria (11.98% SoilA- deviation (n =3). ConA, 8.72% SoilB-ConB, 17.01% CoilC-ConC, and 17.63% SoilD-ConD) and Bacterioidetes (15.39% SoilA- ConA, 9.28% SoilB-ConB, 5.23% CoilC-ConC, and Results 18.04% SoilD-ConD) types. In all cases, except for SoilD, an increase in the percentage ratio of Proteobacteria in the Biodegradation of hydrocarbons consortia compared to that of the soil metapopulations may be observed, and these changes were mainly associated with The results regarding the efficiency of the biological decom- the increased ratio of the Gammaproteobacteria class. The position of selected fractions of hydrocarbons by the consortia analysis of alpha-diversity expressed as the number of OTUs isolated from soils A–D are presented in Fig. 1. The analysis indicated significant differences among all types of collected of samples without the addition of the microbial inoculum soil samples (SoilA-SoilC) (Fig. S2). The addition of diesel oil excluded the abiotic loss of hydrocarbons. The obtained re- to SoilC did not significantly influence the number of OTUs. sults indicated that the consortia differed significantly regard- All consortia, with the exception of ConC, were characterized ing their biodegradation potential. In all studied systems, the by a lower alpha-diversity compared to that of the correspond- most easily biodegradable fraction (> 94%) and the fraction ing soils. Principal coordinate analysis (PCoA) with Bray- characterized by the lowest quantitative diversity after 7 days Curtis dissimilarity (Fig. 3) indicated significant differences of biodegradation (SD = 1.58%) were toluene. The highest between the populations of the studied sites and the cultivated diversity was observed in the alkane group (SD = 9.60%). consortia. The most notable changes between the composition The highest biodegradation potential for this group was ex- of the soil metapopulation and the corresponding consortium hibited by ConC, while ConB displayed the lowest potential. were observed in the cases of ConB and SoilB, whereas the All consortia were characterized by a relatively high potential least significant changes were noted for ConC and ConB. to biodegrade aromatic hydrocarbons, including polycyclic ConD and SoilD exhibit intermediate characteristics between aromatic compounds. Interestingly, consortium ConD exhib- SoilA and SoilC, as well as ConA and ConC. ited a significant, lower level of alkane biodegradation com- pared to that of ConC. The analysis also indicated a lower Predicted functional gene abundance concentration of aromatic and polycyclic aromatic com- pounds in systems inoculated with ConD compared to that The PICRUSt tool was used to predict the functional potential of ConC. Overall, taking the biodegradation of total petroleum of bacterial metapopulations based on the 16S RNA profile. 950 Ann Microbiol (2019) 69:945–955 ConA ConB ConC ConD TPH alkanes toluene naphtalene aromac compounds polyaromac compounds Fig. 1 Biodegradation efficiency of selected fractions of hydrocarbons by environmental consortia in model systems after 7 days of incubation Reference genome coverage for all samples was calculated the lowest amounts were observed in the cases of SoilC and using the weighted Nearest Sequenced Taxon Index score ConC. (NSTI). The NSTI for all samples was in the range of 0.02– 0.06, which indicates a good availability of reference genomes closely related to microorganisms in the sample (Langille et al. Discussion 2013). The total predicted gene abundance divided for the number of OTUs in each sample is presented in Figs. 4 and ABA has been widely considered as a method with a high 5. A relatively high abundance of enzymes participating in application potential due to the natural adaptation of autoch- alkane biodegradation was established in all samples: alde- thonous microorganisms to occupy their native soil environ- hyde dehydrogenase (EC 1.2.1.3) and alcohol dehydrogenase ment. However, the efficiency of this method notably depends (EC 1.1.1.1). In the group of enzymes participating in the on the potential of microorganisms to biodegrade xenobiotics biodegradation of naphthalene, toluene, and polycyclic aro- and proliferate under laboratory conditions (Dott et al. 1989; matic hydrocarbons (PAH), the highest indications were ob- Vecchioli et al. 1990; Hosokawa et al. 2009). The terrestrial served in the case of SoilB. environment is a dynamic, multi-phase system, which results in the diversification of the local structure of autochthonous Microarray analysis microbial metapopulations and, consequently, their metabolic profiles. The analysis of β-diversity based on the Bray-Curtis The results of the semi-quantitative analysis of the presence of dissimilarity of soil samples indicated that the microbial com- copies of the selected genes are presented in Fig. 6.Genes munities of specific sites differ from each other. The highest alkB1 and alkB2 were characterized by the highest frequency distance was observed in the case of SoilC, representing urban of occurrence in SoilD, ConC, and ConD, whereas systems A areas, which may be associated with the co-existence of and B exhibited the highest abundances of genes participating anthropogenic-based selection factors. The assessment of dis- in the degradation pathways of aromatic and polycyclic aro- tances in the Bray-Curtis dissimilarity analysis for SoilA, matic hydrocarbons. In all cases, an increase in the share of SoilC, and SoilD indicated that the changes in the soil popu- analyzed gene copies in cultivated consortia compared to that lation were occurring because the supply of hydrocarbons is in the respective soils was observed. SoilB and ConB exhib- targeted and not accidental. ited the highest numbers of genes with a high abundance in the The Sphingobacteriia class is particularly sensitive, as its soil sample group and consortia group, respectively, whereas percentage ratio was lower in the metapopulation of SoilA, Hydrocarbon residue [%] Ann Microbiol (2019) 69:945–955 951 Fig. 2 Relative changes in bacterial phyla (a) and classes (b) 100% in soils A–D and consortia A–D Acnobacteria 90% 80% Bacteroidetes 70% 60% Cyanobacteria 50% 40% Firmicutes 30% Proteobacteria 20% 10% N/A 0% SoilA ConA SoilB ConB SoilC ConC SoilD ConD Acnobacteria 100% 90% Flavobacteria 80% Sphingobacteria 70% Bacilli 60% Clostridia 50% 40% Alphaproteobacteria 30% Betaproteobacteria 20% Deltaproteobacteria 10% Gammaproteobacteria 0% SoilA ConA SoilB ConB SoilC ConC SoilD ConD N/A SoilB, and SoilD in comparison to SoilC. A decreasing ten- characterized by their high resistance to the respective stress dency of biodiversity in environments characterized by high factors were capable of growth, due to the long-term effect of anthropogenization is also observed, especially in strongly hydrocarbon contaminants. The reduction in biodiversity may contaminated areas (SoilA) in which the lowest number of also be caused by the formation of dead-end during the bio- OTUs was established. This outcome may result from the fact degradation of PAH, which has a toxic impact on part of the that only microorganisms with enzymatic profiles that are soil metapopulation (Ghosal et al. 2016). specialized for the biodegradation of xenobiotics and are The prediction of gene abundance based on 16S rRNA data indicated the important diversification of the soil enzymatic PCo 2 (20%) potential. The amounts of estimated gene copies encoding SoilB enzymes participating in the biodegradation of alkanes—EC 1.2.1.3 (aldehyde dehydrogenase) and EC 1.1.1.1 (alcohol dehydrogenase)—were particularly high in SoilD. This out- ConB come may be associated with the fact that in soils permanently subjected to anthropression (SoilA and SoilB), the microor- ConC ganisms adapted to the decomposition of hydrocarbons are characterized by lower bioavailability due to the dissipation SoilC PCo 1 (41%) SoilD of easily biodegradable hydrocarbons. In soil system type D, ConD SoilA ConA the highest amount of gene copies of alkane 1- monooxygenase (alkB1 and alkB2) was also observed to be much higher than that in the case of SoilC; this outcome may PCo 3 (14%) indicate a rapid adaptation in the microbial community to the higher supply of hydrocarbons. Microbial metapopulations Fig. 3 PCoAwith Bray-Curtis dissimilarity based on the 16S rRNA gene 952 Ann Microbiol (2019) 69:945–955 Fig. 5 Heatmap of enzymatic potential based on predicted gene Fig. 4 Heatmap of enzymatic potential based on predicted gene abundance for naphthalene and PAH biodegradation abundance for alkane and toluene biodegradation toluene-3-monooxygenase pathway in all analyzed soil sys- are characterized by the dynamics of qualitative and quantita- tems, in which phenol 2-monooxygenase is used (EC tive changes due to the changes in environmental factors and 1.14.13.7). In SoilB, there are additionally high indications exposition to toxic compounds (Wyrwas et al. 2013). During for the predictions of EC 1.14.12.11 (toluene 1,2- dioxygenase) and EC 1.3.8.3 (benzylsuccinyl-CoA dehydro- bioremediation processes, the activation of microflora capable of degrading alkanes occurs first, whereas groups capable of genase), indicating the presence of alternative pathways of degrading more complex structures tend to dominate in the long-term (Bento et al. 2005). This phenomenon is also reflected by the analysis of genes responsible for the biodeg- radation of aromatic and polycyclic aromatic hydrocarbons in soil samples. The highest estimated indications of enzymes initiating the pathway for the biological degradation of PAH, EC 1.14.12.12 (naphthalene 1,2-dioxygenase) and EC 1.3.1.29 (naphthalene dihydrodiol dehydrogenase), were pres- ent in SoilA and SoilB. The most important indications for the selected genes participating in the biodegradation of aromatic compounds ndoB, nahG, cat 2, cat 3,and bphC were also noted in such systems. In the case of toluene biodegradation, due to the relatively good representation of data associated with the KEGG, different biodegradation strategies may be distinguished. The prediction indicates the presence of the Fig. 6 Heatmap of gene abundance measured using microarrays Ann Microbiol (2019) 69:945–955 953 biological decomposition—namely dioxygenase mediated ConB and ConC. The cultivation under laboratory conditions pathway and anaerobic toluene pathway with benzyl succinate results in the notable reduction in the number of OTUs. Some intermediate, respectively. The presence of dioxygenase- soil microorganisms are not capable of efficient growth mediated pathway confirms the high indication for the xylE gene in vitro (Pham and Kim 2012). The analysis of the ADP/ in SoilB. (Parales et al. 2008). The results representing the pre- ATP ratio indicated that all consortia in a holistic approach dictions for several Enzyme Commission numbers (EC) are char- are characterized by the ability to proliferate under laboratory acterized by low indications, which may be associated with the conditions using diesel oil as the sole carbon source. The limited availability of annotated reference genomes and the hor- highest indications were noted for ConC and ConD, which izontal gene transfer in bacterial metapopulations mentioned in are reflected by the TPH biodegradation efficiency. A similar the literature (Jiménez et al. 2014). Furthermore, it should be tendency is observed in the case of alkane biodegradation, noted that the relatively high results of representation for SoilB which is associated with the fact that alkanes constitute more and ConB result from the lowest NSTI coefficient among all the than 60% (w/w) of diesel oil (Liang et al. 2005). The biodeg- analyzed samples (0.023 and 0.028, respectively). radation of toluene occurred at a high efficiency in all the The comparison involving the enzymatic potential of inde- analyzed systems. This outcome may result from the fact that pendent samples is therefore semi-quantitative. The use of mi- microorganisms capable of decomposing this compound are croarray analysis overcomes these limitations. The method al- widely distributed in nature. Furthermore, toluene is charac- lows for the direct detection of any selected, defined genes in a terized by a relatively high solubility in water in comparison to metapopulation. However, considering the analysis of the meta- other diesel oil hydrocarbons, making it an easily degradable bolic pathways of hydrocarbon biodegradation and the abun- substrate (Singh and Celin 2010). Predictions involving the dance of enzymes participating in these processes, a complex use of the PICRUSt tool indicated that there is a lack of an analysis seems very challenging to conduct. The analyses of anaerobic toluene pathway with benzyl succinate intermediate genes responsible for the initial stages of biological decomposi- in ConB, as observed in SoilB, which may be associated with tion may be a good solution, which, as confirmed by the present- the improvement of aerobic conditions. Moreover, the de- ed studies, allow the elucidation of the differences in terms of the crease in the number of EC indications for toluene-3- genetic potential of bacterial metapopulations and initial predic- monooxygenase pathway in ConA, ConB, and ConD in com- tion of the biodegradation efficiency of selected hydrocarbon parison to that for the soil samples most likely results from the fractions. reduction in alpha biodiversity. In the case of the biodegrada- The comparison of the metapopulation structure of bacterial tion of polycyclic aromatic hydrocarbons, the highest efficien- cultures (ConA-ConD) with their respective soils (SoilA-SoilD) cy was established in soils with a moderate and high level of indicates significant changes. The increase in the anthropogenization. The long-term supply of hydrocarbon contaminants resulted in the succession of the metapopulation Gammaproteobacteria class in ConA, ConB, and ConC demon- strates that this class possesses preferences to proliferate in a towards the biodegradation of poorly available hydrocarbon hydrocarbon-rich environment. Therefore, it displays a high ap- fractions. This phenomenon has found its confirmation in plication potential for ABA. The lack of significant changes in ConD, in which a higher biodegradation efficiency of aromat- ConD results from the fact that the concentration of the hydrocar- ic and polycyclic aromatic hydrocarbons in comparison to bon substrate in SoilD was at least as high as that in the liquid ConC was observed, whereas the biodegradation potential of cultures. This outcome allows the conclusion that the increase in alkanes was decreased. The decrease in the estimated abun- the Gammaproteobacteria ratio did not result directly from pref- dance for EC 1.14.12.12 (naphthalene 1,2-dioxygenase) in erences to grow in a liquid medium. The increase in the ConA and ConB, in comparison to that for the soil samples, Gammaproteobacteria ratio due to the increased supply of n- may result from the fact that there was a supply of more easily alkanes and cycloalkanes in an aqueous environment was also degradable hydrocarbons in the case of laboratory cultivations described in several reports in the literature (Dubinsky et al. 2013; with the addition of diesel oil and—similar to toluene—from Mason et al. 2014; Ferguson et al. 2017). Furthermore, some the reduction in biodiversity. The dynamics of the metapopu- microorganisms belonging to this class, e.g., Pseudomonas sp., lation change in terms of how hydrocarbon contamination is a are capable of degrading several groups of hydrocarbons: alkanes, targeted process. However, in this field, further studies are cycloalkanes, and aromatic compounds (Sydow et al. 2016). required because of the complexity of the soil environment. Another interesting relation may be observed in the case of Sphingobacteria. This class seems to be particularly sensitive to anthropogenic stress factors. In systems A and D, in which Conclusions the concentration of contaminants in the soil was highest and the deterioration of aerobic conditions could also occur, an The efficiency of ABA strictly depends on the genetic poten- increase in the Sphingobacteria ratio in cultivated consortia tial of soil metapopulations. The process of cultivating autoch- was observed. A different relation is observed in the cases of thonous microorganisms in liquid systems with a selective 954 Ann Microbiol (2019) 69:945–955 DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, factor—diesel oil hydrocarbons as the sole carbon source— Huber T, Dalevi D, Hu P, Andersen GL (2006) Greengenes, a notably contributed to the increase in the genetic application chimera-checked 16S rRNA gene database and workbench compat- potential of the obtained inoculates. The studies indicated that ible with ARB. Appl Environ Microbiol 72:5069–5072. https://doi. soils with a high level of anthropogenization, with long-term org/10.1128/AEM.03006-05 Dott W, Feidieker D, Kämpfer P, Schleibinger H, Strechel S (1989) exposition to a high concentration of petroleum compounds Comparison of autochthonous bacteria and commercially available and from uncontaminated areas, exhibit a hydrocarbon bio- cultures with respect to their effectiveness in fuel oil degradation. J degradation potential. To increase the efficiency of soil treat- Ind Microbiol 4:365–373 ment processes and determine the range of future Dubinsky EA, Conrad ME, Chakraborty R, Bill M, Borglin SE, Hollibaugh JT, Mason OU, Piceno MY, Reid FC, Stringfellow implementations, appropriate tools that allow the evaluation WT, Tom LM, Hazen TC, Andersen GL (2013) Succession of of the presence of genes crucial for the proper metabolic path- hydrocarbon-degrading bacteria in the aftermath of the deepwater ways are necessary. Microarrays are a good alternative for horizon oil spill in the gulf of Mexico. Environ Sci Technol 47: bioinformatic predictions that require costly sequencing pro- 10860–10867. https://doi.org/10.1021/es401676y Ferguson RMW, Gontikaki E, Anderson JA, Witte U (2017) The variable cedures. Due to the spontaneous, dynamic changes that occur influence of dispersant on degradation of oil hydrocarbons in sub- in the metapopulations from the moment of contamination, arctic deep-sea sediments at low temperatures (0–5 °C). Sci Rep 7: the use of microarrays at a semi-quantitative level of evalua- 2253. https://doi.org/10.1038/s41598-017-02475-9 tion seems to be sufficient. Notably, in the case of the presence Ghosal D, Ghosh S, Dutta TK, Ahn Y (2016) Current state of knowledge in microbial degradation of polycyclic aromatic hydrocarbons of microorganisms with specific metabolic preferences, they (PAHs): a review. Front Microbiol 7:1369. https://doi.org/10.3389/ can dominate the system as a result of succession processes fmicb.2016.01369 and efficiently improve the bioremediation efficiency. Hosokawa R, Nagai M, Morikawa M, Okuyama H (2009) Autochthonous bioaugmentation and its possible application to oil Funding This study was funded by the National Science Centre in Poland spills. World J Microbiol Biotechnol 25:1519–1528. https://doi.org/ in the years 2014–2018 with the research project Opus no. 2013/11/B/ 10.1007/s11274-009-0044-0 NZ9/01908. Jiménez DJ, Dini-Andreote F, van Elsas JD (2014) Metataxonomic pro- filing and prediction of functional behaviour of wheat straw degrading microbial consortia. Biotechnol Biofuels 7:92. https:// Data availability The sequencing datasets generated and analyzed during doi.org/10.1186/1754-6834-7-92 the current study are available in the SRA repository, with the identifier Kanehisa M, Goto S (2000) KEGG: Kyoto encyclopedia of genes and SRP145038 (https://www.ncbi.nlm.nih.gov/sra/SRP145038). genomes. Nucleic Acids Res 28:27–30 Karigar CS, Rao SS (2011) Role of microbial enzymes in the bioremedi- Compliance with ethical standards ation of pollutants: a review. Enzyme Res:805187. https://doi.org/ 10.4061/2011/805187 Kumar A, Bisht BS, Joshi VD, Dhewa T (2011) Review on bioremedia- Conflict of interest The authors declare that they have no conflict of tion of polluted environment: a management tool. Int J Environ Sci interest. 1(6):1079–1093 Langille MGI, Zaneveld J, Caporaso JG, McDonald D, Knights D, Reyes Human and animal rights and informed consent This research does not J, Clemente JC, Burkepile DE, Thurber RLV, Knight R, Beiko RG, involve human participants and/or animals; therefore, no informed con- Huttenhower C (2013) Predictive functional profiling of microbial sent is needed. communities using 16S rRNA marker gene sequences. Nat Biotechnol 31:814–821. https://doi.org/10.1038/nbt.2676 Liang F, Lu M, Keener TC, Liu Z, Khang SJ (2005) The organic com- position of diesel particulate matter, diesel fuel and engine oil of a non-road diesel generator. J Environ Monit 7:983–988. https://doi. References org/10.1039/B504728E Macaulay BM, Rees D (2014) Bioremediation of oil spills: a review of Adams GO, Fufeyin PT, Okoro SE, Ehinomen I (2015) Bioremediation, challenges for research advancement. Ann Environ Sci 8:9–37 biostimulation and bioaugmention: a review. Int J Environ Mason OU, Han J, Woyke T, Jansson JK (2014) Single-cell genomics Bioremediat Biodegrad 3:28–39. https://doi.org/10.12691/ijebb-3- reveals features of a Colwellia species that was dominant during the 1-5 Deepwater Horizon oil spill. Front Microbiol 5(332). https://doi.org/ Bento FM, Camargo FA, Okeke BC, Frankenberger WT (2005) 10.3389/fmicb.2014.00332 Comparative bioremediation of soils contaminated with diesel oil Nikolopoulou M, Eickenbusch P, Pasadakis N, Venieri D, Kalogerakis N by natural attenuation, biostimulation and bioaugmentation. (2013) Microcosm evaluation of autochthonous bioaugmentation to Bioresour Technol 96:1049–1055. https://doi.org/10.1016/j. combat marine oil spills. New Biotechnol 30:734–742. https://doi. biortech.2004.09.008 org/10.1016/j.nbt.2013.06.005 Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer Parales RE, Parales JV, Pelletier DA, Ditty JL (2008) Diversity of micro- N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, bial toluene degradation pathways. Adv Appl Microbiol 64:1–73. Smith G, Knight R (2012) Ultra-high-throughput microbial commu- https://doi.org/10.1016/S0065-2164(08)00401-2 nity analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6: Pham VH, Kim J (2012) Cultivation of unculturable soil bacteria. Trends 1621–1624. https://doi.org/10.1038/ismej.2012.8 Biotechnol 30:475–484. https://doi.org/10.1016/j.tibtech.2012.05. Das N, Chandran P (2011) Microbial degradation of petroleum hydrocar- bon contaminants: an overview. Biotechnol Res Int:941810. https:// Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, doi.org/10.4061/2011/941810 Glöckner FO (2013) The SILVA ribosomal RNA gene database Ann Microbiol (2019) 69:945–955 955 project: improved data processing and web-based tools. Nucleic Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JA (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 41:D590–D596. https://doi.org/10.1093/nar/gks1219 Sierra-Garcia I, Alvarez JC, Vasconcellos SP, Souza AP, Neto EV, Acids 35:W71–W74. https://doi.org/10.1093/nar/gkm306 Oliviera VM (2014) New hydrocarbon degradation pathways in Vecchioli GI, Del Panno MT, Painceira MT (1990) Use of selected au- the microbial metagenome from Brazilian petroleum reservoirs. tochthonous soil bacteria to enhanced degradation of hydrocarbons PLoS One 9(2):e90087. https://doi.org/10.1371/journal.pone. in soil. Environ Pollut 67:249–258 Wanger A, Chavez V, Huang RSP, Wahed A, Actor JK, Dasgupta A Singh R, Celin SM (2010) Biodegradation of BTEX (benzene, toluene, (2017) Media for the Clinical Microbiology Laboratory. ethyl benzene and xylene) compounds by bacterial strain under aer- Microbiology and molecular diagnosis in pathology, pp 51–60. obic conditions. J Ecobiotechnol 2:27–32 https://doi.org/10.1016/B978-0-12-805351-5.00004-1 Singh P, Jain R, Srivastava N, Borthakur A, Pal DB, Singh R, Madhav S, Wu M, Dick WA, Li W, Wang X, Yang Q, Wang T, Xu L, Zhang M, Chen Srivastava P, Tiwary D, Mishra PK (2017) Current and emerging L (2016) Bioaugmentation and biostimulation of hydrocarbon deg- trends in bioremediation of petrochemical waste: a review. Crit Rev radation and the microbial community in a petroleum-contaminated Environ Sci Technol 47:155–201. https://doi.org/10.1080/ soil. Int Biodeterior Biodegradation 107:158–164. https://doi.org/ 10643389.2017.1318616 10.1016/j.ibiod.2015.11.019 Sydow M, Owsianiak M, Szczepaniak Z, Framski G, Smets BF, Wyrwas B, Dymaczewski Z, Zgoła-Grześkowiak A, Szymański A, Ławniczak Ł, Lisiecki P, Szulc A, Cyplik P, Chrzanowski Ł Frańska M, Kruszelnicka I, Ginter-Kramarczyk D, Cyplik P, (2016) Evaluating robustness of a diesel-degrading bacterial consor- Ławniczak Ł, Chrzanowski Ł (2013) Biodegradation of Triton X- tium isolated from contaminated soil. New Biotechnol 33:852–859. 100 and its primary metabolites by a bacterial community isolated https://doi.org/10.1016/j.nbt.2016.08.003 from activated sludge. J Environ Manag 128:292–299. https://doi. Szczepaniak Z, Czarny J, Staninska-Pięta J, Lisiecki P, Zgoła- org/10.1016/j.jenvman.2013.05.028 Grześkowiak A, Cyplik P, Chrzanowski Ł, Wolko Ł, Marecik R, Zivelytea V, Valtersb K, Vasareviciusa S (2017) Comparison between Juzwa W, Glazar K, Piotrowska-Cyplik A (2016) Influence of soil passive remediation and bioremediation for the hydrocarbons con- contamination with PAH on microbial community dynamics and taminated soil clean up. Energy Procedia 128:339–344 expression level of genes responsible for biodegradation of PAH and production of rhamnolipids. Environ Sci Pollut Res 23: 23043–23056. https://doi.org/10.1007/s11356-016-7500-9 Ueno A, Ito Y, Yumoto I, Okuyama H (2007) Isolation and characteriza- tion of bacteria from soil contaminated with diesel oil and the pos- Publisher’snote Springer Nature remains neutral with regard to sible use of these in autochthonous bioaugmentation. World J jurisdictional claims in published maps and institutional affiliations. Microbiol Biotechnol 23:1739–1745. https://doi.org/10.1007/ s11274-007-9423-6 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Assessment of soil potential to natural attenuation and autochthonous bioaugmentation using microarray and functional predictions from metagenome profiling

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Springer Journals
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Copyright © 2019 by Università degli studi di Milano
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-019-01486-3
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Abstract

Purpose The use of autochthonous microorganisms for the bioaugmentation of areas contaminated with hydrocarbons has a high potential to overcome the limitations associated with the difficulty of allochthonous microorganisms to adapt. The prediction of bioremediation effects of autochthonous bioaugmentation can be improved by employing the rapid methods of the direct detection of genes crucial to the hydrocarbon biodegradation. This study aimed to evaluate the potential of microflora originating from soils with different levels of anthropogenization for application in autochthonous bioaugmentation by using microarray and functional predictions from metagenome profiling. Methods Analyses based on the modern techniques of molecular biology—DNA microarrays and next-generation sequencing— coupled with the functional predictions of metagenome profiling. Results Studies indicated that the metapopulations of all analyzed stations possess the ability to biodegrade petroleum hydrocarbons. It was established that the long-term supply of hydrocarbons in the areas characterized by strong anthropogenization resulted in increasing the biological decomposition of aromatic and polycyclic aromatic com- pounds. In contrast, areas with a low level of anthropogenization were characterized by a higher potential to decompose aliphatic hydrocarbons. Although alpha-biodiversity decreased when the consortia was isolated and cul- tivated under laboratory conditions with hydrocarbons as the sole carbon source, microbial communities with genetic biodegradation potential increased, which was confirmed by the analysis involving the loss of selected hydrocarbon fractions in aqueous systems. Conclusions The presented studies indicated the vast potential for the application of isolated autochthonous microflora on soils permanently contaminated with hydrocarbons. The prediction of bioremediation effects may be improved by employing the rapid method of the direct detection of genes crucial to the biological decomposition of hydrocarbons, with DNA microarrays developed in the framework of this study. . . . Keywords Autochthonous bioaugmentation Microarray Hydrocarbon biodegradation Biodiversity Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13213-019-01486-3) contains supplementary material, which is available to authorized users. * Jakub Czarny Department of Biochemistry and Biotechnology, Poznan University pubjc@igs.org.pl of Life Sciences, Dojazd 11, 60-632 Poznan, Poland Department of Rare Earths, Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznan, Poland Institute of Forensic Genetics, Al. Mickiewicza 3/4, 85-071 Bydgoszcz, Poland Department Biotechnology and Food Microbiology, Poznan University of Life Sciences, Wojska Polskiego 48, Institute of Food Technology of Plant Origin, Poznan University of 60-627 Poznan, Poland Life Sciences, Wojska Polskiego 31, 60-624 Poznan, Poland 946 Ann Microbiol (2019) 69:945–955 Introduction molecular metagenomics tools that will allow for their assess- ment (Sierra-Garcia et al. 2014). Increased demand for solid fossil fuels, their intense explora- This study aimed to evaluate the potential of microflora orig- tion and distribution result in the unintended release of petro- inating from soils with different levels of anthropogenization and leum hydrocarbons to the natural environment (Adams et al. supply of petroleum hydrocarbons for the biodegradation of se- 2015; Singh et al. 2017; Zivelytea et al. 2017). It is believed lected fractions of hydrocarbons based on the modern techniques that hydrocarbons are among the most widespread contami- of molecular biology—namely DNA microarrays and next- nants in highly industrialized and developing countries generation sequencing coupled with the functional prediction of (Macaulay and Rees 2014). Local contamination of soil may metagenome profiling. Additionally, the ability of consortia to result from accidental leaks during the extraction, refining, proliferate under laboratory conditions and the biodegradation transport, and storage of fossil fuels, as well as during the efficiency of hydrocarbons under model conditions was ana- improper storage of petroleum products in underground tanks, lyzed. This analysis allowed the identification of the diverse en- the destruction of industrial pipelines and the illegal uptake of zymatic capabilities of soil systems and the determination of the fuels (Das and Chandran 2011). Biological methods based on possibility of using the ABA technology in cases of widespread the natural ability of microorganisms to decompose organic contaminations. compounds enzymatically comprise a promising remediation technology for contaminated areas. These methods are an al- ternative for chemical and physical methods and are charac- Methods terized by their low operational costs and limited risk of trans- formation of xenobiotics to more toxic intermediates (Karigar Soil sampling and Rao 2011; Kumar et al. 2011; Macaulay and Rees 2014). Bioaugmentation is a strategy for the biological decomposi- Three main types of study areas were defined: post-industry tion of petroleum hydrocarbons, and this strategy has been areas with a high level of anthropogenization (A), moderate known since the 1970s and is based on the introduction of level of anthropogenization (B), and low level of microbial populations characterized by a high biodegradation anthropogenization under the statutory form of natural protec- potential. This method is particularly promising in cases in tion (C). In each area, a sampling site was established which microorganisms do not possess the proper metabolic (supplementary materials, Table S1)withan area of 5 m× predispositions to biologically decompose petroleum hydro- 5 m. A composite sample was gathered from each site (com- carbons or their ratio in the population is relatively low posed of 10 partial samples of topsoil layer) using a Shelby (Adams et al. 2015). The efficiency of introducing allochtho- tube sampler (2-cm diameter, 10-cm depth). Additionally, a nous microorganisms with high biodegradation potential is model system for soil contamination with a low level of broadly discussed in the literature. It is believed that the lim- anthropogenization was prepared. Approx. 50 g was taken ited ability of the inoculated consortium to adapt to local con- from the composite soil sample, and then, 5 g of diesel oil ditions and the rivalry for the environmental niche with au- (DO) was added and left at room temperature for 2 months. tochthonous microflora are the factors that are responsible for The soils with the addition of DO were labeled type D the long-term reduction in biodegradation efficiency (Ueno (supplementary materials, Table S1). The selected hydrocar- et al. 2007; Macaulay and Rees 2014). Several authors indi- bon concentrations of the soil samples were presented as a cate that the effect of the stimulation of biodegradation effi- supplementary material, Table S2. ciency does not occur or is only short-term and that it is insig- nificant in the removal of hydrocarbons (Bento et al. 2005; Isolation of consortia and preparation Szczepaniak et al. 2016). Furthermore, it is believed that the of the inoculum efficiency of biodegradation processes is correlated, not with biodiversity, but rather with the number of individual micro- Approx. 10 g of soil was taken from each sample, added to organisms, which are capable of decomposing petroleum hy- 90 mL of 0.9% solution of NaCl and shaken for 30 min drocarbons (Wu et al. 2016). A novel method to overcome the (150 rpm). Next, the systems were left for 10 min to allow limitations associated with the adaptation of allochthonous for the sedimentation of mineral particles, and then, 1 mL of microorganisms may be the autochthonous bioaugmentation the water phase was transferred to flasks containing tryptic soy (ABA) technique, which is based on the re-introduction of broth (common nutritional medium to supports the growth of local microflora. To date, there are only a few research studies a wide range of bacteria) (Wanger et al. 2017) with the addi- involving this field (Ueno et al. 2007; Nikolopoulou et al. tion of 2% of glucose. The obtained consortia (ConA-ConD) 2013). Since the efficiency of all bioremediation techniques, were grown for 72 h under aerobic conditions on a shaker including ABA, is correlated with the enzymatic potential of (130 rpm), then centrifuged (10 min, 4000 rpm), and rinsed autochthonous microflora, there is a need to develop three times with 0.9% solution of NaCl, centrifuging each Ann Microbiol (2019) 69:945–955 947 time according to the described procedure. The final inoculum follows: interface temperature 230 °C, scanning interval 0.15 s, was suspended in 0.9% solution of NaCl by normalizing the and scanning range 33–250 m/z. concentration based on optical density OD (600) to the value of 0.6 (Helios Delta Vis, ThermoFisher Scientific Inc., USA). Assessment of viability of consortia Biodegradation of hydrocarbons in model systems Samples were taken from the model system after 24, 48, 72, Biodegradation was conducted in Duran-type flasks with baf- and 168 h of biodegradation. To determine the viability of fles, in model aqueous systems with the addition of diesel oil consortia, a commercially available ADP/ATP Ratio Assay as the sole carbon source. The experimental system was com- Kit (Sigma-Aldrich Inc.) was used. Approx. 10 μLof each posed of 50 mL of mineral medium with microelements sample was transferred to a 96-well plate, and then, the ATP (Na HPO 6.21 g/L, KH PO 2.8 g/L, NaCl 0.5 g/L, NH Cl reagent (prepared in accordance with the manufacturers’ pro- 2 4 2 4 4 1.0 g/L, MgSO ×7H O 0.01 g/L, FeSO ×7H O 0.001 g/L, tocol) was added. The samples were incubated for 60 s at 4 2 4 2 MnSO ×4H O 0.0005 g/L, ZnCl 0.00064 g/L, CaCl × room temperature, and then, the luminescence (RLUa) was 4 2 2 2 6H O 0.0001 g/L, BaCl 0.00006 g/L, CoSO ×7H O measured using a SpectraMax M2e multi-mode plate reader 2 2 4 2 0.000036 g/L, CuSO ×5H O 0.000036 g/L, H BO (Molecular Devices Inc.). The samples were incubated for 4 2 3 3 0.00065 g/L, H MoO 0.005 g/L, EDTA 0.001 g/L, HCl another 10 min, and the luminescence (RLUb) was measured 2 4 37% 0.0146 mL/L), 1 g of diesel oil, and 250 μLofthe again. Next, 5 μL of the ADP Reagent (prepared in accor- microbial inoculum. Biodegradation was conducted for 7 days dance with the manufacturers’ protocol) was added. After at 25 °C under aerobic conditions on a shaker (130 rpm). Each 60 s, the luminescence (RLUc) was measured for the third sample was prepared in six repetitions. Three were used for time. The ADP/ATP ratio was calculated based on the follow- the further analysis of hydrocarbon biodegradation, and three ing formula: were used for the analysis of the viability of consortia. Additionally, three internal controls without the addition of ADP RLUc−RLUb ratio ¼ microorganisms were prepared to exclude the abiotic loss of ATP RLUa hydrocarbons. Analysis of the biodegradation of selected fractions Isolation of DNA and sequencing of hydrocarbons Isolation of DNA After 7 days of the biodegradation process, the samples were subjected to extraction. Approx. 2.5 mL of acetone The isolation of the genetic material from the soil samples and was added to each system and shaken for 10 min the liquid cultures was conducted using the following kits: (150 rpm). Next, 200 μL of internal standards was intro- Genomic Mini AX Soil Spin (A&A biotechnology Inc.) and duced: n-nonane (Sigma-Aldrich Inc.), n-octacosane Genomic Mini AX Bacteria Spin (A&A Biotechnology Inc.), (Restek Inc.), and acenaphthene-d10 (Restek Inc.) with respectively, by following the manufacturers’ recommendations. concentrations of 6 mg/mL, 6 mg/mL, and 1.5 mg/mL, The validation of isolation efficiency was carried out with a respectively, in an acetone/hexane mixture (2:3 v/v). fluorometric method using a Qubit 3.0 apparatus and Qubit™ Then, 25 mL of hexane (Poch Inc.) was added, and the dsDNA HS Assay Kit (ThermoFisher Scientific Inc.). samples were shaken again for 60 min (150 rpm). The samples were stabilized for 30 min until the phases were separated, and 2 mL of acetone (Poch Inc.) was added PCR amplification and sequencing dropwise to remove any emulsions. The prepared extracts were diluted 25-fold in hexane and subjected to GC-MS Universal prokaryotic primers 515F-806R were used to am- analysis. plify the V4 region of 16S rRNA (Caporaso et al. 2012). The The GC-MS analysis was carried out using a Shimadzu 17A PCR reaction was carried out in a volume of 25 μL(5 μL gas chromatograph coupled with a QP5000 mass spectrometer microbial template genomic DNA, 5 μL of each primer, equipped with a Rxi-5MS COLUMN (Restek Inc.). The follow- 2.5 μL of PCR-grade water (ThermoFisher Scientific Inc.), ing separation conditions were employed: carrier gas (helium) and 12.5 μL of PCR Master Mix with the Taq polymerase flow 1.1 mL/min, injection port temperature 250 °C, splitless (ThermoFisher Scientific Inc.). The following PCR reaction injection mode, injection volume 1.0 μL, and column tempera- conditions were employed: initial denaturation 95 °C for ture program 40 °C held for 1 min, ramped at rate of 15 °C/min 3 min; 35 cycles 1 min at 95 °C, 30 s at 52 °C, and 1 min at to 300 °C, and held for 7 min. Detection conditions were as 72 °C; and final extension at 72 °C for 10 min. The amplicons 948 Ann Microbiol (2019) 69:945–955 were purified using Clean-Up columns (A&A Biotechnology the following sequence: 5′-C12-AMINO-(dT)18-ACG TAC Inc.) and then used for the construction of libraries. GTA CGT ACG TAC GTA CGT-Cy5-3′. Sequencing was conducted using a MiSeq (Illumina Inc., All four sub-matrices are printed on the glass using the CA) apparatus with a MiSeq Reagent Kit v2 (2 × 250 bp) same system. Upon application of the probes, the binding (Illumina Inc.). Details regarding the preparation of libraries process was conducted for 12 h at room temperature and hu- were described in a previous study (Szczepaniak et al. 2016). midity < 30%, and then, the systems are rinsed two times with a 0.1% solution of sodium dodecyl sulfate and two more times Analysis of data after sequencing with water. Raw data in the FASTQ format were imported to the CLC DNA extraction and amplification Genomics Workbench 8.5 software with the CLC Microbial Genomics Module 1.2 (Qiagen Inc.). The reads were DNA was isolated from the soil and batch cultures as de- demultiplexed, and paired ends were merged (mismatch scribed in the previous section. cost = 2, min score = 8, Gap cost = 3, max unaligned end mis- PCR amplification was conducted in two multiplex reac- matches = 5). Next, the primer sequences were trimmed (qual- tions. Two different fragments were amplified for each gene, ity limit = 0.05, ambiguous limit = N), and the identification and one was amplified for each multiplex reaction; they were and elimination of the chimeric reads were conducted. The detected using complementary probes (supplementary output data were clustered independently based on two refer- materials, Table S3). Multiplex 1 and 2 PCR amplifications ence databases: SILVA v119 (Quast et al. 2013)and were conducted in a 50 μL volume using specific starters GreenGenes 13.5 (DeSantis et al. 2006) at a 97% probability (supplementary materials, Table S3) at the following condi- level of open taxonomic units (OTUs). Based on the merged tions: 1× PCR buffer, MgCl 2.5 mM, dNTPs 250 μM(A&A abundance table (clustered against SILVA v119), the alpha- Biotechnology Inc.), BSA FractionV 0.16 mg/mL (Sigma- biodiversity (number of OTUs) and beta-biodiversity (Bray- Aldrich Inc.), Glycerol (10% w/v), 5 U TaqPol (A&A Curtis PCoA) factors were determined. Biotechnology Inc.), starters based on the Table S3 (supple- To predict the gene content from each OTUs table con- mentary materials), and 10 μL of DNA. The following tem- structed against GreenGenes 13.5, the Phylogenetic perature profile was used for the reaction: initial denaturation Investigation of Communities by Reconstruction of at 95 °C for 3 min, and then, 35 cycles denaturation at 94 °C Unobserved States (PICRUSt) tool was used (Langille et al. for 1 min, annealing 60 °C for 1 min, elongation 72 °C for 2013). The output data were a collection involving the abun- 1 min, and final elongation 60 °C for 15 min. Two studied dance of key functional orthologues (KO) in each sample. samples are subjected to amplification—namely negative con- Based on the Kyoto Encyclopedia of Genes and Genomes trol for DNA isolation and positive control for PCR (KEGG), the KO in hydrocarbon biodegradation was selected, amplification. which allowed the prediction of the functional pathways The amplification products of Multiplex 1 and 2 were (Kanehisa and Goto 2000). Finally, the data were grouped pooled and purified from the starters and nucleotides using based on the EC classification. To compare the samples, the the precipitation method on the clean-up columns (A&A results were normalized by dividing the total predicted gene Biotechnology Inc.). The purified products were rinsed from abundance for the number of OTUs in each sample. the column using a volume of 50 μL. Each test study includes the analysis of the positive control, the negative control, and Microarrays the studied sample (two replications). Array preparation Matrix analysis Four round areas with a diameter of 9 mm (sub-matrices) were A60-μL aliquot of the reaction mixture was applied to the separated on SUPEROXY glass using 0.25-mm foil. separated submatrices for the positive control, the negative Oligonucleotides with a sequence complementary to the am- control, and the studied samples. The reaction mixture plified sequences were applied to fixed places on the four contained the following: 1× SBE Buffer, 0.75 μMof 7- areas by the contact method using SpotBot 3 Microarrayer propargylamino-7-deaza-2′,3′-dideoxyadenosine-5′ (Arrayit Inc.). Probe sequences were established using thiphosphate-6-FAM, 5-propargylamino-2 ′,3 ′- Primer3 Plus software (supplementary materials, Table S3) dideoxycytidine-5′thiphosphate-6-FAM, 7-propargylamino- (Untergasser et al. 2007). On the 5′ end, each probe would 7-deaza-2′3′dideoxyguanosine-5′triphosphate6-FAM, 5- possess a C12-AMINO modification and 18dT sequence pre- propargylamino-2′,3′-dideoxyuridine-5′triphosphate-6-FAM, ceding the correct oligonucleotide sequence. In each 5-propargylamino-2′,3′-dideoxyuridine-5′triphosphate-6- submatrix, a control for matrix preparation is applied with FAM (Jena Bioscience Inc.), 5 U DynaSeq DNA Polymerase Ann Microbiol (2019) 69:945–955 949 (Finnzyme Inc.), and 20 μL of the purified PCR product. The hydrocarbons (TPH) into account, the highest efficiency of the matrix was covered with foil for coating PCR plates. biological decomposition of diesel oil was observed in the Amplification was conducted in a thermocycler equipped with case of ConC. a microscopic glass amplification unit (Dual Flat Block GeneAmp PCR System 9700 Life Technologies Inc., Assessment of viability of consortia Mastercycler Nexus Flat Eppendorf Inc.) at the following con- ditions: 30 cycles denaturation at 94 °C for 1 min, annealing/ To estimate the viability and proliferation of consortia, changes in elongation 50 °C for 1 min, and over temperature 50 °C. the ADP/ATP ratio (supplementary materials, Fig. S1) were de- After the termination of the reaction, the matrix was termined. All consortia after 24 h of biodegradation entered a rinsed using a High Throughput Wash Station (Arrayit phase of exponential growth. The lowest ADP/ATP ratio was Inc.) twice for 2 min in deionized water and dried by noted in the case of ConB. centrifugation for 1 min at 500g in a Microarray High- Speed Centrifuge (Arrayit Inc.). Metapopulation analysis The matrices were analyzed using a GenePix 4300A scan- ner (Molecular Devices Inc.) in an ozone-free cell at excitation Taxonomic identification using the SILVA v119 database and emission wavelengths of 492 and 517 nm, respectively, based on the V4 region of 16S rRNA allowed for a summary for 6-FAM and 625 and 670 nm, respectively, for Cy-5 (spot- detection of 5 phyla, 10 classes, 23 orders, 61 families, 152 ting control). genera, and 389 species of microorganisms. The structure of the metapopulations of the soils and consortia cultivated under Statistical analysis laboratory conditions is presented in Fig. 2. Proteobacteria was the dominant phylum in all samples. The presence of To evaluate the significance of the differences among the an- Cyanobacteria was observed only in SoilB (0.47%). Taking alyzed systems, a nonparametric Kruskal–Wallis test was the comparison of the taxonomic composition of soils and the employed. Mann-Whitney test was used to test differences corresponding consortia into account, the highest differences between groups. All data represent the mean and the standard were observed in the case of Proteobacteria (11.98% SoilA- deviation (n =3). ConA, 8.72% SoilB-ConB, 17.01% CoilC-ConC, and 17.63% SoilD-ConD) and Bacterioidetes (15.39% SoilA- ConA, 9.28% SoilB-ConB, 5.23% CoilC-ConC, and Results 18.04% SoilD-ConD) types. In all cases, except for SoilD, an increase in the percentage ratio of Proteobacteria in the Biodegradation of hydrocarbons consortia compared to that of the soil metapopulations may be observed, and these changes were mainly associated with The results regarding the efficiency of the biological decom- the increased ratio of the Gammaproteobacteria class. The position of selected fractions of hydrocarbons by the consortia analysis of alpha-diversity expressed as the number of OTUs isolated from soils A–D are presented in Fig. 1. The analysis indicated significant differences among all types of collected of samples without the addition of the microbial inoculum soil samples (SoilA-SoilC) (Fig. S2). The addition of diesel oil excluded the abiotic loss of hydrocarbons. The obtained re- to SoilC did not significantly influence the number of OTUs. sults indicated that the consortia differed significantly regard- All consortia, with the exception of ConC, were characterized ing their biodegradation potential. In all studied systems, the by a lower alpha-diversity compared to that of the correspond- most easily biodegradable fraction (> 94%) and the fraction ing soils. Principal coordinate analysis (PCoA) with Bray- characterized by the lowest quantitative diversity after 7 days Curtis dissimilarity (Fig. 3) indicated significant differences of biodegradation (SD = 1.58%) were toluene. The highest between the populations of the studied sites and the cultivated diversity was observed in the alkane group (SD = 9.60%). consortia. The most notable changes between the composition The highest biodegradation potential for this group was ex- of the soil metapopulation and the corresponding consortium hibited by ConC, while ConB displayed the lowest potential. were observed in the cases of ConB and SoilB, whereas the All consortia were characterized by a relatively high potential least significant changes were noted for ConC and ConB. to biodegrade aromatic hydrocarbons, including polycyclic ConD and SoilD exhibit intermediate characteristics between aromatic compounds. Interestingly, consortium ConD exhib- SoilA and SoilC, as well as ConA and ConC. ited a significant, lower level of alkane biodegradation com- pared to that of ConC. The analysis also indicated a lower Predicted functional gene abundance concentration of aromatic and polycyclic aromatic com- pounds in systems inoculated with ConD compared to that The PICRUSt tool was used to predict the functional potential of ConC. Overall, taking the biodegradation of total petroleum of bacterial metapopulations based on the 16S RNA profile. 950 Ann Microbiol (2019) 69:945–955 ConA ConB ConC ConD TPH alkanes toluene naphtalene aromac compounds polyaromac compounds Fig. 1 Biodegradation efficiency of selected fractions of hydrocarbons by environmental consortia in model systems after 7 days of incubation Reference genome coverage for all samples was calculated the lowest amounts were observed in the cases of SoilC and using the weighted Nearest Sequenced Taxon Index score ConC. (NSTI). The NSTI for all samples was in the range of 0.02– 0.06, which indicates a good availability of reference genomes closely related to microorganisms in the sample (Langille et al. Discussion 2013). The total predicted gene abundance divided for the number of OTUs in each sample is presented in Figs. 4 and ABA has been widely considered as a method with a high 5. A relatively high abundance of enzymes participating in application potential due to the natural adaptation of autoch- alkane biodegradation was established in all samples: alde- thonous microorganisms to occupy their native soil environ- hyde dehydrogenase (EC 1.2.1.3) and alcohol dehydrogenase ment. However, the efficiency of this method notably depends (EC 1.1.1.1). In the group of enzymes participating in the on the potential of microorganisms to biodegrade xenobiotics biodegradation of naphthalene, toluene, and polycyclic aro- and proliferate under laboratory conditions (Dott et al. 1989; matic hydrocarbons (PAH), the highest indications were ob- Vecchioli et al. 1990; Hosokawa et al. 2009). The terrestrial served in the case of SoilB. environment is a dynamic, multi-phase system, which results in the diversification of the local structure of autochthonous Microarray analysis microbial metapopulations and, consequently, their metabolic profiles. The analysis of β-diversity based on the Bray-Curtis The results of the semi-quantitative analysis of the presence of dissimilarity of soil samples indicated that the microbial com- copies of the selected genes are presented in Fig. 6.Genes munities of specific sites differ from each other. The highest alkB1 and alkB2 were characterized by the highest frequency distance was observed in the case of SoilC, representing urban of occurrence in SoilD, ConC, and ConD, whereas systems A areas, which may be associated with the co-existence of and B exhibited the highest abundances of genes participating anthropogenic-based selection factors. The assessment of dis- in the degradation pathways of aromatic and polycyclic aro- tances in the Bray-Curtis dissimilarity analysis for SoilA, matic hydrocarbons. In all cases, an increase in the share of SoilC, and SoilD indicated that the changes in the soil popu- analyzed gene copies in cultivated consortia compared to that lation were occurring because the supply of hydrocarbons is in the respective soils was observed. SoilB and ConB exhib- targeted and not accidental. ited the highest numbers of genes with a high abundance in the The Sphingobacteriia class is particularly sensitive, as its soil sample group and consortia group, respectively, whereas percentage ratio was lower in the metapopulation of SoilA, Hydrocarbon residue [%] Ann Microbiol (2019) 69:945–955 951 Fig. 2 Relative changes in bacterial phyla (a) and classes (b) 100% in soils A–D and consortia A–D Acnobacteria 90% 80% Bacteroidetes 70% 60% Cyanobacteria 50% 40% Firmicutes 30% Proteobacteria 20% 10% N/A 0% SoilA ConA SoilB ConB SoilC ConC SoilD ConD Acnobacteria 100% 90% Flavobacteria 80% Sphingobacteria 70% Bacilli 60% Clostridia 50% 40% Alphaproteobacteria 30% Betaproteobacteria 20% Deltaproteobacteria 10% Gammaproteobacteria 0% SoilA ConA SoilB ConB SoilC ConC SoilD ConD N/A SoilB, and SoilD in comparison to SoilC. A decreasing ten- characterized by their high resistance to the respective stress dency of biodiversity in environments characterized by high factors were capable of growth, due to the long-term effect of anthropogenization is also observed, especially in strongly hydrocarbon contaminants. The reduction in biodiversity may contaminated areas (SoilA) in which the lowest number of also be caused by the formation of dead-end during the bio- OTUs was established. This outcome may result from the fact degradation of PAH, which has a toxic impact on part of the that only microorganisms with enzymatic profiles that are soil metapopulation (Ghosal et al. 2016). specialized for the biodegradation of xenobiotics and are The prediction of gene abundance based on 16S rRNA data indicated the important diversification of the soil enzymatic PCo 2 (20%) potential. The amounts of estimated gene copies encoding SoilB enzymes participating in the biodegradation of alkanes—EC 1.2.1.3 (aldehyde dehydrogenase) and EC 1.1.1.1 (alcohol dehydrogenase)—were particularly high in SoilD. This out- ConB come may be associated with the fact that in soils permanently subjected to anthropression (SoilA and SoilB), the microor- ConC ganisms adapted to the decomposition of hydrocarbons are characterized by lower bioavailability due to the dissipation SoilC PCo 1 (41%) SoilD of easily biodegradable hydrocarbons. In soil system type D, ConD SoilA ConA the highest amount of gene copies of alkane 1- monooxygenase (alkB1 and alkB2) was also observed to be much higher than that in the case of SoilC; this outcome may PCo 3 (14%) indicate a rapid adaptation in the microbial community to the higher supply of hydrocarbons. Microbial metapopulations Fig. 3 PCoAwith Bray-Curtis dissimilarity based on the 16S rRNA gene 952 Ann Microbiol (2019) 69:945–955 Fig. 5 Heatmap of enzymatic potential based on predicted gene Fig. 4 Heatmap of enzymatic potential based on predicted gene abundance for naphthalene and PAH biodegradation abundance for alkane and toluene biodegradation toluene-3-monooxygenase pathway in all analyzed soil sys- are characterized by the dynamics of qualitative and quantita- tems, in which phenol 2-monooxygenase is used (EC tive changes due to the changes in environmental factors and 1.14.13.7). In SoilB, there are additionally high indications exposition to toxic compounds (Wyrwas et al. 2013). During for the predictions of EC 1.14.12.11 (toluene 1,2- dioxygenase) and EC 1.3.8.3 (benzylsuccinyl-CoA dehydro- bioremediation processes, the activation of microflora capable of degrading alkanes occurs first, whereas groups capable of genase), indicating the presence of alternative pathways of degrading more complex structures tend to dominate in the long-term (Bento et al. 2005). This phenomenon is also reflected by the analysis of genes responsible for the biodeg- radation of aromatic and polycyclic aromatic hydrocarbons in soil samples. The highest estimated indications of enzymes initiating the pathway for the biological degradation of PAH, EC 1.14.12.12 (naphthalene 1,2-dioxygenase) and EC 1.3.1.29 (naphthalene dihydrodiol dehydrogenase), were pres- ent in SoilA and SoilB. The most important indications for the selected genes participating in the biodegradation of aromatic compounds ndoB, nahG, cat 2, cat 3,and bphC were also noted in such systems. In the case of toluene biodegradation, due to the relatively good representation of data associated with the KEGG, different biodegradation strategies may be distinguished. The prediction indicates the presence of the Fig. 6 Heatmap of gene abundance measured using microarrays Ann Microbiol (2019) 69:945–955 953 biological decomposition—namely dioxygenase mediated ConB and ConC. The cultivation under laboratory conditions pathway and anaerobic toluene pathway with benzyl succinate results in the notable reduction in the number of OTUs. Some intermediate, respectively. The presence of dioxygenase- soil microorganisms are not capable of efficient growth mediated pathway confirms the high indication for the xylE gene in vitro (Pham and Kim 2012). The analysis of the ADP/ in SoilB. (Parales et al. 2008). The results representing the pre- ATP ratio indicated that all consortia in a holistic approach dictions for several Enzyme Commission numbers (EC) are char- are characterized by the ability to proliferate under laboratory acterized by low indications, which may be associated with the conditions using diesel oil as the sole carbon source. The limited availability of annotated reference genomes and the hor- highest indications were noted for ConC and ConD, which izontal gene transfer in bacterial metapopulations mentioned in are reflected by the TPH biodegradation efficiency. A similar the literature (Jiménez et al. 2014). Furthermore, it should be tendency is observed in the case of alkane biodegradation, noted that the relatively high results of representation for SoilB which is associated with the fact that alkanes constitute more and ConB result from the lowest NSTI coefficient among all the than 60% (w/w) of diesel oil (Liang et al. 2005). The biodeg- analyzed samples (0.023 and 0.028, respectively). radation of toluene occurred at a high efficiency in all the The comparison involving the enzymatic potential of inde- analyzed systems. This outcome may result from the fact that pendent samples is therefore semi-quantitative. The use of mi- microorganisms capable of decomposing this compound are croarray analysis overcomes these limitations. The method al- widely distributed in nature. Furthermore, toluene is charac- lows for the direct detection of any selected, defined genes in a terized by a relatively high solubility in water in comparison to metapopulation. However, considering the analysis of the meta- other diesel oil hydrocarbons, making it an easily degradable bolic pathways of hydrocarbon biodegradation and the abun- substrate (Singh and Celin 2010). Predictions involving the dance of enzymes participating in these processes, a complex use of the PICRUSt tool indicated that there is a lack of an analysis seems very challenging to conduct. The analyses of anaerobic toluene pathway with benzyl succinate intermediate genes responsible for the initial stages of biological decomposi- in ConB, as observed in SoilB, which may be associated with tion may be a good solution, which, as confirmed by the present- the improvement of aerobic conditions. Moreover, the de- ed studies, allow the elucidation of the differences in terms of the crease in the number of EC indications for toluene-3- genetic potential of bacterial metapopulations and initial predic- monooxygenase pathway in ConA, ConB, and ConD in com- tion of the biodegradation efficiency of selected hydrocarbon parison to that for the soil samples most likely results from the fractions. reduction in alpha biodiversity. In the case of the biodegrada- The comparison of the metapopulation structure of bacterial tion of polycyclic aromatic hydrocarbons, the highest efficien- cultures (ConA-ConD) with their respective soils (SoilA-SoilD) cy was established in soils with a moderate and high level of indicates significant changes. The increase in the anthropogenization. The long-term supply of hydrocarbon contaminants resulted in the succession of the metapopulation Gammaproteobacteria class in ConA, ConB, and ConC demon- strates that this class possesses preferences to proliferate in a towards the biodegradation of poorly available hydrocarbon hydrocarbon-rich environment. Therefore, it displays a high ap- fractions. This phenomenon has found its confirmation in plication potential for ABA. The lack of significant changes in ConD, in which a higher biodegradation efficiency of aromat- ConD results from the fact that the concentration of the hydrocar- ic and polycyclic aromatic hydrocarbons in comparison to bon substrate in SoilD was at least as high as that in the liquid ConC was observed, whereas the biodegradation potential of cultures. This outcome allows the conclusion that the increase in alkanes was decreased. The decrease in the estimated abun- the Gammaproteobacteria ratio did not result directly from pref- dance for EC 1.14.12.12 (naphthalene 1,2-dioxygenase) in erences to grow in a liquid medium. The increase in the ConA and ConB, in comparison to that for the soil samples, Gammaproteobacteria ratio due to the increased supply of n- may result from the fact that there was a supply of more easily alkanes and cycloalkanes in an aqueous environment was also degradable hydrocarbons in the case of laboratory cultivations described in several reports in the literature (Dubinsky et al. 2013; with the addition of diesel oil and—similar to toluene—from Mason et al. 2014; Ferguson et al. 2017). Furthermore, some the reduction in biodiversity. The dynamics of the metapopu- microorganisms belonging to this class, e.g., Pseudomonas sp., lation change in terms of how hydrocarbon contamination is a are capable of degrading several groups of hydrocarbons: alkanes, targeted process. However, in this field, further studies are cycloalkanes, and aromatic compounds (Sydow et al. 2016). required because of the complexity of the soil environment. Another interesting relation may be observed in the case of Sphingobacteria. This class seems to be particularly sensitive to anthropogenic stress factors. In systems A and D, in which Conclusions the concentration of contaminants in the soil was highest and the deterioration of aerobic conditions could also occur, an The efficiency of ABA strictly depends on the genetic poten- increase in the Sphingobacteria ratio in cultivated consortia tial of soil metapopulations. The process of cultivating autoch- was observed. A different relation is observed in the cases of thonous microorganisms in liquid systems with a selective 954 Ann Microbiol (2019) 69:945–955 DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, factor—diesel oil hydrocarbons as the sole carbon source— Huber T, Dalevi D, Hu P, Andersen GL (2006) Greengenes, a notably contributed to the increase in the genetic application chimera-checked 16S rRNA gene database and workbench compat- potential of the obtained inoculates. The studies indicated that ible with ARB. Appl Environ Microbiol 72:5069–5072. https://doi. soils with a high level of anthropogenization, with long-term org/10.1128/AEM.03006-05 Dott W, Feidieker D, Kämpfer P, Schleibinger H, Strechel S (1989) exposition to a high concentration of petroleum compounds Comparison of autochthonous bacteria and commercially available and from uncontaminated areas, exhibit a hydrocarbon bio- cultures with respect to their effectiveness in fuel oil degradation. J degradation potential. To increase the efficiency of soil treat- Ind Microbiol 4:365–373 ment processes and determine the range of future Dubinsky EA, Conrad ME, Chakraborty R, Bill M, Borglin SE, Hollibaugh JT, Mason OU, Piceno MY, Reid FC, Stringfellow implementations, appropriate tools that allow the evaluation WT, Tom LM, Hazen TC, Andersen GL (2013) Succession of of the presence of genes crucial for the proper metabolic path- hydrocarbon-degrading bacteria in the aftermath of the deepwater ways are necessary. Microarrays are a good alternative for horizon oil spill in the gulf of Mexico. Environ Sci Technol 47: bioinformatic predictions that require costly sequencing pro- 10860–10867. https://doi.org/10.1021/es401676y Ferguson RMW, Gontikaki E, Anderson JA, Witte U (2017) The variable cedures. Due to the spontaneous, dynamic changes that occur influence of dispersant on degradation of oil hydrocarbons in sub- in the metapopulations from the moment of contamination, arctic deep-sea sediments at low temperatures (0–5 °C). Sci Rep 7: the use of microarrays at a semi-quantitative level of evalua- 2253. https://doi.org/10.1038/s41598-017-02475-9 tion seems to be sufficient. Notably, in the case of the presence Ghosal D, Ghosh S, Dutta TK, Ahn Y (2016) Current state of knowledge in microbial degradation of polycyclic aromatic hydrocarbons of microorganisms with specific metabolic preferences, they (PAHs): a review. Front Microbiol 7:1369. https://doi.org/10.3389/ can dominate the system as a result of succession processes fmicb.2016.01369 and efficiently improve the bioremediation efficiency. Hosokawa R, Nagai M, Morikawa M, Okuyama H (2009) Autochthonous bioaugmentation and its possible application to oil Funding This study was funded by the National Science Centre in Poland spills. World J Microbiol Biotechnol 25:1519–1528. https://doi.org/ in the years 2014–2018 with the research project Opus no. 2013/11/B/ 10.1007/s11274-009-0044-0 NZ9/01908. Jiménez DJ, Dini-Andreote F, van Elsas JD (2014) Metataxonomic pro- filing and prediction of functional behaviour of wheat straw degrading microbial consortia. Biotechnol Biofuels 7:92. https:// Data availability The sequencing datasets generated and analyzed during doi.org/10.1186/1754-6834-7-92 the current study are available in the SRA repository, with the identifier Kanehisa M, Goto S (2000) KEGG: Kyoto encyclopedia of genes and SRP145038 (https://www.ncbi.nlm.nih.gov/sra/SRP145038). genomes. Nucleic Acids Res 28:27–30 Karigar CS, Rao SS (2011) Role of microbial enzymes in the bioremedi- Compliance with ethical standards ation of pollutants: a review. Enzyme Res:805187. https://doi.org/ 10.4061/2011/805187 Kumar A, Bisht BS, Joshi VD, Dhewa T (2011) Review on bioremedia- Conflict of interest The authors declare that they have no conflict of tion of polluted environment: a management tool. Int J Environ Sci interest. 1(6):1079–1093 Langille MGI, Zaneveld J, Caporaso JG, McDonald D, Knights D, Reyes Human and animal rights and informed consent This research does not J, Clemente JC, Burkepile DE, Thurber RLV, Knight R, Beiko RG, involve human participants and/or animals; therefore, no informed con- Huttenhower C (2013) Predictive functional profiling of microbial sent is needed. communities using 16S rRNA marker gene sequences. Nat Biotechnol 31:814–821. https://doi.org/10.1038/nbt.2676 Liang F, Lu M, Keener TC, Liu Z, Khang SJ (2005) The organic com- position of diesel particulate matter, diesel fuel and engine oil of a non-road diesel generator. J Environ Monit 7:983–988. https://doi. References org/10.1039/B504728E Macaulay BM, Rees D (2014) Bioremediation of oil spills: a review of Adams GO, Fufeyin PT, Okoro SE, Ehinomen I (2015) Bioremediation, challenges for research advancement. Ann Environ Sci 8:9–37 biostimulation and bioaugmention: a review. Int J Environ Mason OU, Han J, Woyke T, Jansson JK (2014) Single-cell genomics Bioremediat Biodegrad 3:28–39. https://doi.org/10.12691/ijebb-3- reveals features of a Colwellia species that was dominant during the 1-5 Deepwater Horizon oil spill. Front Microbiol 5(332). https://doi.org/ Bento FM, Camargo FA, Okeke BC, Frankenberger WT (2005) 10.3389/fmicb.2014.00332 Comparative bioremediation of soils contaminated with diesel oil Nikolopoulou M, Eickenbusch P, Pasadakis N, Venieri D, Kalogerakis N by natural attenuation, biostimulation and bioaugmentation. (2013) Microcosm evaluation of autochthonous bioaugmentation to Bioresour Technol 96:1049–1055. https://doi.org/10.1016/j. combat marine oil spills. New Biotechnol 30:734–742. https://doi. biortech.2004.09.008 org/10.1016/j.nbt.2013.06.005 Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer Parales RE, Parales JV, Pelletier DA, Ditty JL (2008) Diversity of micro- N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, bial toluene degradation pathways. Adv Appl Microbiol 64:1–73. Smith G, Knight R (2012) Ultra-high-throughput microbial commu- https://doi.org/10.1016/S0065-2164(08)00401-2 nity analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6: Pham VH, Kim J (2012) Cultivation of unculturable soil bacteria. Trends 1621–1624. https://doi.org/10.1038/ismej.2012.8 Biotechnol 30:475–484. https://doi.org/10.1016/j.tibtech.2012.05. Das N, Chandran P (2011) Microbial degradation of petroleum hydrocar- bon contaminants: an overview. Biotechnol Res Int:941810. https:// Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, doi.org/10.4061/2011/941810 Glöckner FO (2013) The SILVA ribosomal RNA gene database Ann Microbiol (2019) 69:945–955 955 project: improved data processing and web-based tools. Nucleic Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JA (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 41:D590–D596. https://doi.org/10.1093/nar/gks1219 Sierra-Garcia I, Alvarez JC, Vasconcellos SP, Souza AP, Neto EV, Acids 35:W71–W74. https://doi.org/10.1093/nar/gkm306 Oliviera VM (2014) New hydrocarbon degradation pathways in Vecchioli GI, Del Panno MT, Painceira MT (1990) Use of selected au- the microbial metagenome from Brazilian petroleum reservoirs. tochthonous soil bacteria to enhanced degradation of hydrocarbons PLoS One 9(2):e90087. https://doi.org/10.1371/journal.pone. in soil. Environ Pollut 67:249–258 Wanger A, Chavez V, Huang RSP, Wahed A, Actor JK, Dasgupta A Singh R, Celin SM (2010) Biodegradation of BTEX (benzene, toluene, (2017) Media for the Clinical Microbiology Laboratory. ethyl benzene and xylene) compounds by bacterial strain under aer- Microbiology and molecular diagnosis in pathology, pp 51–60. obic conditions. J Ecobiotechnol 2:27–32 https://doi.org/10.1016/B978-0-12-805351-5.00004-1 Singh P, Jain R, Srivastava N, Borthakur A, Pal DB, Singh R, Madhav S, Wu M, Dick WA, Li W, Wang X, Yang Q, Wang T, Xu L, Zhang M, Chen Srivastava P, Tiwary D, Mishra PK (2017) Current and emerging L (2016) Bioaugmentation and biostimulation of hydrocarbon deg- trends in bioremediation of petrochemical waste: a review. Crit Rev radation and the microbial community in a petroleum-contaminated Environ Sci Technol 47:155–201. https://doi.org/10.1080/ soil. Int Biodeterior Biodegradation 107:158–164. https://doi.org/ 10643389.2017.1318616 10.1016/j.ibiod.2015.11.019 Sydow M, Owsianiak M, Szczepaniak Z, Framski G, Smets BF, Wyrwas B, Dymaczewski Z, Zgoła-Grześkowiak A, Szymański A, Ławniczak Ł, Lisiecki P, Szulc A, Cyplik P, Chrzanowski Ł Frańska M, Kruszelnicka I, Ginter-Kramarczyk D, Cyplik P, (2016) Evaluating robustness of a diesel-degrading bacterial consor- Ławniczak Ł, Chrzanowski Ł (2013) Biodegradation of Triton X- tium isolated from contaminated soil. New Biotechnol 33:852–859. 100 and its primary metabolites by a bacterial community isolated https://doi.org/10.1016/j.nbt.2016.08.003 from activated sludge. J Environ Manag 128:292–299. https://doi. Szczepaniak Z, Czarny J, Staninska-Pięta J, Lisiecki P, Zgoła- org/10.1016/j.jenvman.2013.05.028 Grześkowiak A, Cyplik P, Chrzanowski Ł, Wolko Ł, Marecik R, Zivelytea V, Valtersb K, Vasareviciusa S (2017) Comparison between Juzwa W, Glazar K, Piotrowska-Cyplik A (2016) Influence of soil passive remediation and bioremediation for the hydrocarbons con- contamination with PAH on microbial community dynamics and taminated soil clean up. Energy Procedia 128:339–344 expression level of genes responsible for biodegradation of PAH and production of rhamnolipids. Environ Sci Pollut Res 23: 23043–23056. https://doi.org/10.1007/s11356-016-7500-9 Ueno A, Ito Y, Yumoto I, Okuyama H (2007) Isolation and characteriza- tion of bacteria from soil contaminated with diesel oil and the pos- Publisher’snote Springer Nature remains neutral with regard to sible use of these in autochthonous bioaugmentation. World J jurisdictional claims in published maps and institutional affiliations. Microbiol Biotechnol 23:1739–1745. https://doi.org/10.1007/ s11274-007-9423-6

Journal

Annals of Microbiology – Springer Journals

Published: Jun 3, 2019

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