Bacteria degrading both n-alkanes and aromatic hydrocarbons are prevalent in soils.

This study was undertaken to determine the distribution of soil bacteria capable of utilizing both n-alkanes and aromatic hydrocarbons. These microorganisms have not been comprehensively investigated so far. Ten contaminated (4046-43,861 mg of total petroleum hydrocarbons (TPH) kg-1 of dry weight of soil) and five unpolluted (320-2754 mg TPH kg-1 of dry weight of soil) soil samples from temperate, arid, and Alpine soils were subjected to isolation of degraders with extended preferences and shotgun metagenomic sequencing (selected samples). The applied approach allowed to reveal that (a) these bacteria can be isolated from pristine and polluted soils, and (b) the distribution of alkane monooxygenase (alkB) and aromatic ring hydroxylating dioxygenases (ARHDs) encoding genes is not associated with the contamination presence. Some alkB and ARHD genes shared the same taxonomic affiliation; they were most often linked with the Rhodococcus, Pseudomonas, and Mycolicibacterium genera. Moreover, these taxa together with the Paeniglutamicibacter genus constituted the most numerous groups among 132 culturable strains growing in the presence of both n-alkanes and aromatic hydrocarbons. All those results indicate (a) the prevalence of the hydrocarbon degraders with extended preferences and (b) the potential of uncontaminated soil as a source of hydrocarbon degraders applied for bioremediation purposes.

Oil-Contaminated Soil Remediation with Biodegradation by Autochthonous Microorganisms and Phytoremediation by Maize (Zea mays).

Biological methods are currently the most commonly used methods for removing hazardous substances from land. This research work focuses on the remediation of oil-contaminated land. The biodegradation of aliphatic hydrocarbons and PAHs as a result of inoculation with biopreparations B1 and B2 was investigated. Biopreparation B1 was developed on the basis of autochthonous bacteria, consisting of strains Dietzia sp. IN118, Gordonia sp. IN101, Mycolicibacterium frederiksbergense IN53, Rhodococcus erythropolis IN119, Rhodococcus globerulus IN113 and Raoultella sp. IN109, whereas biopreparation B2 was enriched with fungi, such as Aspergillus sydowii, Aspergillus versicolor, Candida sp., Cladosporium halotolerans, Penicillium chrysogenum. As a result of biodegradation tests conducted under ex situ conditions for soil inoculated with biopreparation B1, the concentrations of TPH and PAH were reduced by 31.85% and 27.41%, respectively. Soil inoculation with biopreparation B2 turned out to be more effective, as a result of which the concentration of TPH was reduced by 41.67% and PAH by 34.73%. Another issue was the phytoremediation of the pre-treated G6-3B2 soil with the use of Zea mays. The tests were carried out in three systems (system 1-soil G6-3B2 + Zea mays; system 2-soil G6-3B2 + biopreparation B2 + Zea mays; system 3-soil G6-3B2 + biopreparation B2 with γ-PGA + Zea mays) for 6 months. The highest degree of TPH and PAH reduction was obtained in system 3, amounting to 65.35% and 60.80%, respectively. The lowest phytoremediation efficiency was recorded in the non-inoculated system 1, where the concentration of TPH was reduced by 22.80% and PAH by 18.48%. Toxicological tests carried out using PhytotoxkitTM, OstracodtoxkitTM and Microtox® Solid Phase tests confirmed the effectiveness of remediation procedures and showed a correlation between the concentration of petroleum hydrocarbons in the soil and its toxicity. The results obtained during the research indicate the great potential of bioremediation practices with the use of microbial biopreparations and Zea mays in the treatment of soils contaminated with petroleum hydrocarbons.

Evaluation of the Effectiveness of the Biopreparation in Combination with the Polymer γ-PGA for the Biodegradation of Petroleum Contaminants in Soil.

Biodegradation is a method of effectively removing petroleum hydrocarbons from the natural environment. This research focuses on the biodegradation of aliphatic hydrocarbons, monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and all three xylene isomers (BTEX) and polycyclic aromatic hydrocarbons (PAHs) as a result of soil inoculation with a biopreparation A1 based on autochthonous microorganisms and a biopreparation A1 with the addition of γ-PGA. The research used biopreparation A1 made of the following strains: Dietzia sp. IN133, Gordonia sp. IN138 Mycolicibacterium frederiksbergense IN53, Rhodococcus erythropolis IN119, Rhodococcus sp. IN136 and Pseudomonas sp. IN132. The experiments were carried out in laboratory conditions (microbiological tests, respirometric tests, and in semi-technical conditions (ex-situ prism method). The biodegradation efficiency was assessed on the basis of respirometric tests, chromatographic analyses and toxicological tests. As a result of inoculation of AB soil with the biopreparation A1 within 6 months, a reduction of total petroleum hydrocarbons (TPH) (66.03%), BTEX (80.08%) and PAHs (38.86%) was achieved and its toxicity was reduced. Inoculation of AB soil with the biopreparation A1 with the addition of γ-PGA reduced the concentration of TPH, BTEX and PAHs by 79.21%, 90.19%, and 51.18%, respectively, and reduced its toxicity. The conducted research has shown that the addition of γ-PGA affects the efficiency of the biodegradation process of petroleum pollutants, increasing the degree of TPH biodegradation by 13.18%, BTEX by 10.11% and PAHs by 12.32% compared to pure biopreparation A1.

Assessment of the Suitability of Melilotus officinalis for Phytoremediation of Soil Contaminated with Petroleum Hydrocarbons (TPH and PAH), Zn, Pb and Cd Based on Toxicological Tests.

The article presents issues related to the possibility of using toxicological tests as a tool to monitor the progress of soil treatment contaminated with petroleum substances (TPH, PAH), Zn, Pb and Cd in bio-phytoremediation processes. In order to reduce the high content of petroleum pollutants (TPH = 56,371 mg kg-1 dry mass, PAH = 139.3 mg kg-1 dry mass), the technology of stepwise soil treatment was applied, including basic bioremediation and inoculation with biopreparations based of indigenous non-pathogenic species of bacteria, fungi and yeasts. As a result of basic bioremediation in laboratory conditions (ex-situ method), the reduction of petroleum pollutants TPH by 33.9% and PAH by 9.5% was achieved. The introduction of inoculation with biopraparation-1 prepared on the basis of non-pathogenic species of indigenous bacteria made it possible to reduce the TPH content by 86.3%, PAH by 40.3%. The use of a biopreparation-1 enriched with indigenous non-pathogenic species of fungi and yeasts in the third series of inoculation increased to an increase in the degree of biodegradation of aliphatic hydrocarbons with long carbon chains and PAH by a further 28.9%. In the next stage of soil treatment after biodegradation processes, which was characterized by an increased content of heavy metals (Zn, Pb, Cd) and naphthalene, chrysene, benzo(a)anthracene and benzo(ghi)perylene belonging to polycyclic aromatic hydrocarbons, phytoremediation with the use of Melilotus officinalis was applied. After the six-month phytoremediation process, the following was achieved: Zn content by 25.1%, Pb by 27.9%, Cd by 23.2% and TPH by 42.2% and PAH by 49.9%. The rate of removal of individual groups of hydrocarbons was in the decreasing order: C12-C18 > C6-C12 > C18-C25 > C25-C36. PAHs tended to be removed in the following order: chrysene > naphthalene > benzo(a)anthracene > benzo(ghi)perylene. The TF and BCF coefficients were calculated to assess the capacity of M. officinalis to accumulate metal in tissues, uptake from soil and transfer from roots to shoots. The values of TF translocation coefficients were, respectively, for Zn (0.44), Pb (0.12), Cd (0.40). The calculated BCF concentration factors (BCFroots > BCFshoots) show that heavy metals taken up by M. officinalis are mainly accumulated in the root tissues in the following order Zn > Pb > Cd, revealing a poor metal translocation from the root to the shoots. This process was carried out in laboratory conditions for a period of 6 months. The process of phytoremediation of contaminated soil using M. officinalis assisted with fertilization was monitored by means of toxicological tests: Microtox, Ostracodtoxkit FTM, MARA and PhytotoxkitTM. The performed phytotoxicity tests have indicated variable sensitivity of the tested plants on contaminants occurring in the studied soils, following the sequence: Lepidium sativum < Sorghum saccharatum < Sinapis alba. The sensitivity of toxicological tests was comparable and increased in the order: MARA < Ostracodtoxkit FTM < Microtox. The results of the toxicological monitoring as a function of the time of soil treatment, together with chemical analyses determining the content of toxicants in soil and biomass M. officinalis, clearly confirmed the effectiveness of the applied concept of bioremediation of soils contaminated with zinc, lead and cadmium in the presence of petroleum hydrocarbons.

Application of Festuca arundinacea in phytoremediation of soils contaminated with Pb, Ni, Cd and petroleum hydrocarbons.

Phytoremediation is a promising "green technique" used to purify contaminated soils. The performed phytoremediation experiments assisted by the fertilization process involving pots of F.arundinacea grown on soils with diverse concentrations and types of contaminations produced the following decreased percentages after 6 months: Pb (25.4-34.1%), Ni (18.7-23.8%), Cd (26.3-46.7%), TPH (49.4-60.1%). Primarily, TPH biodegradation was occurring as a result of basic bioremediation stimulated by adding optimal volumes of biogenic substances and corrections in the soil reaction, while phytoremediation improved this process by 17.4 - 23.1%. The highest drop in a range of 45.6 - 55.5% was recorded for the group of C12-C18 hydrocarbons, with the lowest one for C25-C36, amounting to 9.1-17.4%. Translocation factor values were: TF<1 and ranged, respectively, for: Pb (0.46-0.53), Ni (0.29-0.33), and Cd (0.21-0.25), which indicate that heavy metals absorbed by Festuca arundinacea they mainly accumulated in the root of the tissue in descending order: Cd

Hydrocarbon Removal by Two Differently Developed Microbial Inoculants and Comparing Their Actions with Biostimulation Treatment.

Bioremediation of soils polluted with petroleum compounds is a widely accepted environmental technology. We compared the effects of biostimulation and bioaugmentation of soil historically contaminated with aliphatic and polycyclic aromatic hydrocarbons. The studied bioaugmentation treatments comprised of the introduction of differently developed microbial inoculants, namely: an isolated hydrocarbon-degrading community C1 (undefined-consisting of randomly chosen degraders) and a mixed culture C2 (consisting of seven strains with well-characterized enhanced hydrocarbon-degrading capabilities). Sixty days of remedial treatments resulted in a substantial decrease in total aliphatic hydrocarbon content; however, the action of both inoculants gave a significantly better effect than nutrient amendments (a 69.7% decrease for C1 and 86.8% for C2 vs. 34.9% for biostimulation). The bioaugmentation resulted also in PAH removal, and, again, C2 degraded contaminants more efficiently than C1 (reductions of 85.2% and 64.5%, respectively), while biostimulation itself gave no significant results. Various bioassays applying different organisms (the bacterium Vibrio fischeri, the plants Sorghum saccharatum, Lepidium sativum, and Sinapis alba, and the ostracod Heterocypris incongruens) and Ames test were used to assess, respectively, potential toxicity and mutagenicity risk after bioremediation. Each treatment improved soil quality, however only bioaugmentation with the C2 treatment decreased both toxicity and mutagenicity most efficiently. Illumina high-throughput sequencing revealed the lack of (C1) or limited (C2) ability of the introduced degraders to sustain competition from indigenous microbiota after a 60-day bioremediation process. Thus, bioaugmentation with the bacterial mixed culture C2, made up of identified, hydrocarbon-degrading strains, is clearly a better option for bioremediation purposes when compared to other treatments.

Assessment of Biodegradation Efficiency of Polychlorinated Biphenyls (PCBs) and Petroleum Hydrocarbons (TPH) in Soil Using Three Individual Bacterial Strains and Their Mixed Culture.

Biodegradation is one of the most effective and profitable methods for the elimination of toxic polychlorinated biphenyls (PCBs) and total petroleum hydrocarbons (TPH) from the environment. In this study, aerobic degradation of the mentioned pollutants by bacterial strains Mycolicibacterium frederiksbergense IN53, Rhodococcus erythropolis IN129, and Rhodococcus sp. IN306 and mixed culture M1 developed based on those strains at 1:1:1 ratio was analyzed. The effectiveness of individual strains and of the mixed culture was assessed based on carried out respirometric tests and chromatographic analyses. The Rhodococcus sp. IN306 turned out most effective in terms of 18 PCB congeners biodegradation (54.4%). The biodegradation index was decreasing with an increasing number of chlorine atoms in a molecule. Instead, the Mycolicobacterium frederiksbergense IN53 was the best TPH degrader (37.2%). In a sterile soil, contaminated with PCBs and TPH, the highest biodegradation effectiveness was obtained using inoculation with mixed culture M1, which allowed to reduce both the PCBs (51.8%) and TPH (34.6%) content. The PCBs and TPH biodegradation capacity of the defined mixed culture M1 was verified ex-situ with prism method in a non-sterile soil polluted with aged petroleum hydrocarbons (TPH) and spent transformer oil (PCBs). After inoculation with mixed culture M1, the PCBs were reduced during 6 months by 84.5% and TPH by 70.8% as well as soil toxicity was decreased.

Changes in toxicity during in situ bioremediation of weathered drill wastes contaminated with petroleum hydrocarbons.

Bioremediation of weathered drill wastes severely contaminated with total petroleum hydrocarbons (TPH) (90,000-170,000 mg kg(-1)) and BTEX (51.2-95.5 mg kg(-1)) to soil standards was achieved over a 3-year period in three phases: initial remediation, basic bioremediation and inoculation with a biopreparation. Fourteen non-pathogenic indigenous bacteria species belonging mainly to the Actinomycetales were identified and shown to be able to degrade 63-75% of nC(9)-nC(20), 36-51% of nC(21)-nC(36), 36% of BTEX and 20% of PAHs (polycyclic aromatic hydrocarbons). Addition of five non-pathogenic fungi species to the bacterial consortium allowed degradation of 69-89% of nC(9)-nC(20), 47-80% of nC(21)-nC(36), 76% of BTEX, and 68% of PAHs. Microtox, Ostacodtoxkit, Phytotoxkit and Ames tests indicated that changes in toxicity were not connected with the decrease in TPH contents, possibly due to the formation of toxic indirect metabolites during bioremediation. No toxicity was found in the soil after bioremediation.

Optimisation research of petroleum hydrocarbon biodegradation in weathered drilling wastes from waste pits.

The aim of this article is to discuss the problem of drilling waste remediation. Analyses and research showed that material stored in waste pits could be classified as soil with a high level of petroleum impurities (total petroleum hydrocarbons [TPH] = 102,417-132,472 mg kg(-1) dry mass). While preparing the complex technology of soil decontamination (which included primary reclamation, basic bioremediation and inoculation with biopreparations based on indigenous bacteria and fungi), laboratory tests indicated the use of an ex-situ method was fundamental. Remediation was controlled with a chromatographic method of qualitative and quantitative determination of petroleum hydrocarbons. Based on analytical data, there was the possibility to determine the effectiveness of consecutive purifying phases. Laboratory tests, following 135 days of basic bioremediation stimulated by optimum conditions to activate the growth of indigenous micro-organisms, resulted in a decrease in the TPH content, which was in the range of 52.3-72.5%. The next phase of soil decontamination lasted 135 days and involved the use of inoculation with biopreparations based on indigenous micro-organisms and fungi. This process enabled a TPH decrease of 93.8- 94.3%. Laboratory biodegradation research was done with the use of the biomarker C30-17α(H)21ß(H)-hopane to normalize analyte (TPH, Σn-C8-n-C22 and Σn-C23-n-C36) concentrations. The calculated first-order biodegradation constants enable estimation of the purification stage dynamics and the effectiveness of the applied biopreparations. Furthermore, they represent the biodegradation degree of individual n-alkanes in subsequent stages of the soil purification process.