Co-contamination of soil with hydrocarbons and heavy metals has higher toxicity in the environment than soils with a single contaminant. This is known to lead to the difficulties in soil remediation (Liu et al. 2017). For the remediation of such soils, phytoremediation is considered a gentle and cost-effective method. Nevertheless, the phytoremediation of mixed contaminants is poorly understood, and more details about this process are needed. It has been demonstrated that the use of plants themselves for simultaneous removal of hydrocarbons and heavy metals from soil is very often insufficient. Thus, improvement of the phytoremediation process by supporting this approach with plant growth-promoting bacteria or by the addition of fertilizers is proposed. Nonetheless, such improvements have been poorly studied for soils co-contaminated with hydrocarbons and heavy metals.
In the present study, we analyzed the effect of introducing rhizospheric Pseudomonas qingdaonensis ZCR6 strain and/or the addition of meat and bone meal (MBM) into the soil that is historically co-contaminated with hydrocarbons and heavy metals on the phytoremediation process using Zea mays. The ZCR6 strain was chosen because of its potential to promote plant growth through a wide range of mechanisms, such as the production of IAA, ACC deaminase, siderophores, and ammonia and the solubilization of phosphate, under laboratory conditions. Additionally, this strain was able to degrade petroleum hydrocarbons, showed surface active properties and was resistant to Cd, Zn, and Cu (2022). In our experiment, we did not observe the promoting effect of the living cells of ZCR6 strain on the growth of Z. mays or the removal of hydrocarbons and heavy metals from the contaminated soil. The biomass of roots and shoots of the maize grown in the soils treated with the live ZCR6 strain (BL and BL+MBM) did not differ significantly compared to the biomass of plants grown in soils treated with the thermally inactivated ZCR6 strain (BTI and BTI+MBM) or water (W and W+MBM). Such a situation was also reported by Liu et al. (2021), who studied the effect of the Lelliottia jeotgali MR2 strain on the growth of Miscanthus floridulus (Lab.) and efficiency of the phytoremediation of Cd-contaminated soil. Even though the MR2 strain presented high Cd tolerance and exhibited the high potential of PGP traits in biochemical tests (production of IAA, siderophore and ACCD, N2 fixation, P and K solubilization), it did not significantly promote M. floridulus (Lab.) growth in the pot experiment and had no significant effect on the efficiency of the phytoremediation process, even though the soil contained only one type of contamination. The authors explained this by the fact that the survival of the MR2 endophyte was low in the soil. It is well known that one of the most important factors influencing the efficiency of bioaugmentation and bacteria-supported phytoremediation is the survival of the introduced bacteria after soil inoculation (Płociniczak et al. 2017) and the ability of inoculants to colonize plant roots (de Souza et al. 2015). In our experiment we used the ZCR6 strain isolated from rhizosphere of maize and it survived in the soil and after an initial reduction in its number was able to colonize the maize roots for the entire experimental period. In such a situation, the lack of effect from the strain could be caused by the fact that the introduced strain did not exhibit the same activity in the soil as under laboratory conditions (Boon et al. 2002). The effect that a particular bacterium has on a plant growth depends mainly on the soil characteristics, such as soil type, nutrient pool, soil moisture, autochthonous microorganism number and diversity, and toxic compound concentrations (de Souza et al. 2015). Therefore, apart from monitoring the survival of inoculants, it is important to analyze their activity in contaminated environments (Festa et al. 2016). In our experiment, quantification of the expression level of genes encoding proteins involved in plant growth promotion was thus performed. For this purpose we tracked the sequences encoding several PGP mechanisms existing in the genome of the ZCR6 strain. Nevertheless, we only detected the expression of the acdS gene, encoding ACC deaminase, which was significantly higher in soils not treated with MBM than in soils supplemented with fertilizer. Since ACCD activity plays a significant role in plants exposed to stress conditions (Ptaszek et al. 2020), a lower expression level of the acdS gene in soils in which plants did not survive is understandable. On the other hand, the lack of differences in the level of expression of this gene between BL, BTI and W soils on Day 20 and the occurrence of higher number of its transcripts in soils BTI and W, compared to soil BL on Day 40 supports the hypothesis that this strain did not activate the mechanism of plant growth promotion in soil during the experimental period.
In the current experiment, a rifampicin-resistant mutant of the ZCR6 strain additionally carrying the pMP4655 plasmid vector with constitutive expression of the egfp gene was used. In our previous bioaugmentation experiments we often observed a gradual decrease in the number of introduced strains, and yet we still observed the effect of enhancing the process initiated when the strain was detected in the soil in high amounts. We hypothesized that the introduced strains transited to the VBNC (viable but nonculturable) state (Su et al. 2019). Here, we observed a decrease in the number of rifampicin-resistant cells of the ZCR6 strain while such an intensive decrease was not observed in the number of cells carrying the egfp gene; a result that confirms our published findings (Ptaszek et al. 2020) that the introduced strains, after being introduced into the soil, enter the VBNC state and are not able to grow under laboratory conditions.
The addition of the thermal-inactivated cells of the ZCR6 strain resulted in significantly higher hydrocarbon removal from soil (BTI) on Day 20 compared to soil treated with live cells of this strain (BL). Bacterial dead biomass, being an additional source of energy and carbon, might serve as fast but short time acting biofertilizer leading to increased activity of autochthonous hydrocarbon-degrading microorganisms (Liang et al. 2019); therefore, higher TPH removal efficiency was observed in soil biostimulated with necromass of the ZCR6 strain than bioaugmented with non-active in soil living cells of the ZCR6 strain. However, this effect was temporary because on Day 40, no significant differences in soil TPH content were observed between the soil BTI and soils BL and W. Introduced live cells of the P. qingdaonensis ZCR6, although they survived in the soil during the entire experimental period, they did not contribute to the increased removal of hydrocarbons from the soil. It was reported that microbial degraders often underperform in a soil environment compared with under laboratory conditions due to their inability to cope with various environmental stressors (e.g., competition, predation, temperature, aeration, nutrient starvation, moisture, etc.) (Bodor et al. 2021). It is also worth mentioning that among the genes involved in hydrocarbon degradation, only the CYP153 gene was expressed, which surprisingly, was at a significantly higher level in all soils not enriched with MBM than in soils supplemented with this fertilizer, although significantly higher TPH removal was estimated in MBM soils. Additionally, because the alkB, alkH, C120 and C230 genes were expressed in the studied soils, we hypothesize that the degradation of hydrocarbons could have occurred with the participation of enzymes characteristic of autochthonous soil microorganisms, which have not been tested in this study, but could be revealed in planned metatranscriptomic study.
The efficiency of removing hydrocarbons from soil could be affected by the presence of heavy metals by changing the surface properties of hydrocarbon-degrading microorganisms or by interfering with microbial enzymes involved in the degradation of hydrocarbons. In the first case, heavy metal ions can be attracted to negatively charged microorganisms, and at the beginning, they occupy adsorption areas of hydrocarbons on the microbial surface. Afterward, as the metal cation concentration on the surface increases, the microorganisms charge is gradually neutralized, which leads to a decrease in the hydrophilicity of their cells and promotes hydrocarbon adsorption (Liu et al. 2017). In the second case, heavy metals present in soil at high concentrations can compete with macronutrients (e.g., Mg2+, Ca2+) used as coenzymes; additionally, they can mask catalytically active groups of enzymes decomposing hydrocarbons or combine with sulfhydryl groups of proteins, thus inhibiting the activity of these enzymes (Karaca et al. 2010; Guo et al. 2010). The combined presence of organic and inorganic pollutants in the soil may also affect the phytoremediation process due to their interactions with plants and their rhizospheric microorganisms (Zhang et al. 2011). Interactions among multiple pollutants bring more challenges to plant growth or even survival; therefore, the appropriate intensification of phytoremediation helps to overcome these limitations. In addition to the introduction of plant growth-promoting bacteria, the amendment of soil with fertilizers can significantly increase plant biomass production and soil microbial activity (Shtangeeva et al. 2004; Cao et al. 2022); therefore, in our experiment, we studied the impact of meat and bone meal addition on phytoremediation efficiency. Nevertheless, enrichment of soil co-contaminated with petroleum hydrocarbons (PH) and heavy metals (HM) with MBM had a negative effect on the growth of maize. In all soils enriched with MBM, plants were able to grow only for 10 days after bacterial inoculation, and the biomass of both their roots and shoots was significantly lower at that time compared to plants from soil not enriched with MBM. Additionally, their physiological condition, determined on Day 10 of the experiment based on the pigment content, was significantly worse than the condition of plants from BL, BTI and W soil. They had a significantly lower content of chlorophyll, which reflected their worse photosynthesis ability and nutritional status (Liang et al. 2017; Zulkarnaini et al. 2019), and a significantly higher content of anthocyanins synthesized, inter alia, under stress caused by the presence of heavy metals (Gould 2004). All plants grown in soils supplemented with MBM had significantly higher NBI values than plants from soils not supplemented with this fertilizer, which was related to the increased nitrogen content in the soil after MBM supplementation. Despite a better supply of nitrogen, these plants were in a worse condition, and after Day 10 of the experiment, their death was noted, which was probably related to the lowering of the soil pH after adding meat and bone meal. Because the solubility and mobility of heavy metals in soil increases with a decrease in the pH value (Chuan et al. 1996; Shrestha et al. 2019), we can hypothesize that a sudden drop in pH and the associated sudden increase in the bioavailability of heavy metals for plants caused the stress conditions and led to plant dieback. Our results contradict the observations made by Liu et al. (2019), who reported that during a bioremediation experiment of soil contaminated with diesel oil, the soil remained neutral or slightly alkaline, regardless of the MBM concentration used. They also demonstrated the great potential of meat and bone meal to be used as biostimulation agents to enhance diesel oil degradation in contaminated soil. In their laboratory experiment, oil degradation was fast during the first 28 days (>77% of the initial hydrocarbon content was degraded), and then the removal rate slowed and finally reached >90% after 100 days of the experiment. The positive effect of the MBM on the degradation rate of hydrocarbon pollutants in the soil was also confirmed in our experiment. We observed a significantly higher TPH removal in all soils enriched with MBM on both 20 and 40 days of the experiment compared to soils not enriched with fertilizer. We also reported a fast hydrocarbon degradation rate during the first 20 days of the experiment (mean TPH loss was 32%), followed by no further hydrocarbon removal in the next 20 days. MBM releases nutrients over a long time period, which has earlier enhanced the degradation of organic contaminants in mild climatic conditions (e.g. Liu et al., 2019; Sun et al., 2018). As in Liu et al. (2019), we found that the stimulation was particularly evident in early phase remediation. It thus seems that the addition of slow-releasing nutrients to PH contaminated soil is a superior strategy compared to phytoremediation, providing that the goal is to decrease peak levels quickly. However, based on our results, phytoremediation of co-contaminated soils should not be started simultaneously with MBM addition. Instead, in co-contaminated soils, planting Z. mays a few months after adding MBM could lead to optimal remediation. Then, MBM would stimulate microbial activity and enhance PH degradation, and plants would take up HM without suffering from resource competition and potential toxic degradation products of PH.
In the study of Mondini et al. (2008), the amendment of arable soils with MBM caused an overall increase in the activity of soil microbial biomass (determined on the basis of the hydrolysis of FDA), indicating an enhancement capacity of the soil for element cycling and an improvement of its quality. An increase in FDA hydrolysis also directly indicates an enhanced ability of the soil to degrade and transform organic substrates and pollutants (Sánchez-Monedero et al. 2008), which was confirmed in our experiment. We noted significantly higher hydrocarbon degradation in all soils enriched with MBM, in which significantly higher FDA hydrolysis was obtained. This result also indicated that hydrocarbon removal from soil co-contaminated with PH and HM supplemented with MBM resulted from the enhanced activity of autochthonous microorganisms. This is in agreement with results from our previous studies on the bioaugmentation of soil contaminated only with hydrocarbons in which TPH loss was also noted after the death of the introduced strains Rhodococcus erythropolis CD 130 and CD 167, inoculated as a single strain or as a mixture (Pacwa-Płociniczak et al. 2020).
In our experiment we noted a potential of maize to accumulate Cd, Zn and Cu from the soil in the presence of petroleum hydrocarbons, with the highest accumulation capacity for Zn. The addition of both living and thermally inactivated cells of the ZCR6 strain did not affect the phytoextraction of Cd and Cu, whereas enhanced accumulation of Zn in the roots of maize was observed in soils BL and BTI compared to soil W. Nevertheless, the translocation factor calculated for all tested HMs was not higher than 1, as maize is not hyperaccumulating plant. Similar results also were observed in other studies on phytoextraction of heavy metals from co-contaminated soils using Z. mays (Lin et al. 2008; Zhang et al. 2009b; Hechmi et al. 2013; Nwosu et al. 2018). The main advantage of maize in phytoremediation studies on co-contaminated soils is its tolerance to PH and limited HM uptake capacity. The large size of the roots and thus the extensive rhizosphere zone in which the rhizodegradation of PH takes place, as well as the large biomass accumulating metals make this plant useful in the treatment of co-contaminated areas.
The total concentration of Zn accumulated in maize plants (roots+shoots) varied between treatments and was 2221±293; 2072±234 and 1631±457 mg kg -1 d.w. of plants in the BL, BTI and W treatments, respectively, wherein Zn mostly accumulated in the roots. Gheju and Stelescu (2013) also demonstrated the ability of Z. mays to extract Zn from soils with concentrations of Zn ranging from 64 to 1800 mg kg-1 of plant tissue. They observed that the metal concentration in maize tissues increased in conjunction with the Zn concentration in soil. Additionally, they reported that the application of an ethylene-diamine-tetraacetace (EDTA) chelating agent significantly enhanced Zn uptake and its translocation in maize, thus increasing the Zn phytoextraction potential of this plant. Nevertheless, their studies were conducted with soil artificially contaminated with Zn, which was not co-contaminated with hydrocarbons.