Bioremediation, or the use of microorganisms to detoxify or remove contaminants, has great potential for the remediation of contaminated soils [1
]. Bioremediation can be performed through: (i) natural attenuation, or the natural process of contaminant degradation; (ii) biostimulation, or the modification of the environmental conditions to stimulate the biodegradation ability of indigenous microorganisms; and (iii) bioaugmentation, or the introduction of exogenous microorganisms with the capacity to degrade the target contaminants [4
Industrial soils are most frequently affected by the presence of more than one contaminant, thus hindering the application of biological remediation techniques. For instance, in petroleum contaminated soils, aliphatic and aromatic hydrocarbons [including polycyclic aromatic hydrocarbons (PAHs)] are often mixed [7
]. Among petroleum-derived contaminants, PAHs are of particular concern since they can seriously affect human health [8
Rhizoremediation (i.e., the use of plants and their associated microorganisms to remediate contaminated soils, usually as a result of the stimulation of the catalytic activities of soil microorganisms by plant roots) has great potential for the remediation of organically-contaminated soils [9
], owing to the fact that plant roots emit exudates that provide nutrients and energy for rhizobacteria, which makes rhizospheric microbial communities more abundant and active than those in bulk soil [11
]. In particular, rhizoremediation has been suggested to be the primary mechanism responsible for hydrocarbon degradation in soil [13
]. Actually, some root exudates, such as oxalic acid and citric acid, have the ability to desorb PAHs, thus facilitating their degradation by soil bacterial populations [7
On the other hand, bacterial consortia with the metabolic ability to degrade organic contaminants are often added to contaminated soils in a process called bioaugmentation [5
]. In addition to contaminant-degrading bacterial strains, plant growth-promoting rhizobacteria and bacterial endophytes (i.e., strains isolated from the interior of plant tissues) have demonstrated their potential for phytoremediation, owing to their ability to stimulate plant growth and/or protect plants against contaminant toxicity through several mechanisms [15
]. Thus, it has been reported [17
] that endophyte inoculation can improve the physiological status of Festuca rubra
plants by increasing the content of carotenoids, chlorophylls and the Fv/Fm ratio (an estimate of the photosynthetic efficiency of photosystem II) by 69, 65 and 37%, respectively, while also enhancing the values of a variety of microbial indicators of soil health.
Furthermore, organic amendments, such as animal manure and compost, are recurrently used to improve soil physicochemical (e.g., porosity, aeration, water holding capacity, structural stability, nutrient availability) and biological (e.g., microbial biomass and activity) properties [18
], as well as to promote plant colonization and growth, during biological remediation processes. Composting organic amendments can minimize chemical and especially biological risks, such as, for instance, the presence of potential human pathogens [20
An often-mentioned paradigm within the soil remediation field is that “the ultimate goal of any soil remediation process must be not only to reduce the concentration of the target contaminants but, most importantly, to also improve soil health” [4
]. In this respect, many physicochemical methods of soil remediation are known to cause a negative impact on soil health. As a matter of fact, some of them have been tagged as more damaging to the soil ecosystem than the contaminants themselves [4
]. Indeed, many soil remediation methods actively reduce the concentration of the contaminants at the expense of negatively affecting the integrity of the soil ecosystem, i.e., soil health. Moreover, during the biodegradation of organic contaminants, highly toxic intermediate transformation products can be produced, leading to adverse and frequently unknown consequences for soil health [4
]. Unfortunately, most soil remediation works only aim at reducing the concentration of the target contaminants below regulatory limits, most of which have been established from tests that lack the required level of ecological relevance.
Accordingly, relevant indicators of soil health must always be included in remediation monitoring programs aimed at evaluating the effectiveness of the applied treatments (effectiveness in terms of soil health improvement). As compared to physicochemical properties, microbial parameters are increasingly being used as indicators of soil health, owing to their sensitivity, fast response, ecological relevance, and capacity to provide information that integrates different environmental factors [24
In this study, we aimed to remediate an organically-contaminated industrial soil using a combination of assisted rhizoremediation and bioaugmentation. We hypothesized that the combination of approaches such as (i) assisted rhizoremediation with Brassica napus plants, and (ii) bioaugmentation with a bacterial consortium with the metabolic ability to degrade hydrocarbons and promote the growth of plants, would both reduce the concentration of soil contaminants and improve soil health. For that reason, we assessed the effectiveness of the applied remediation treatments in terms of both (i) reduction in contaminant concentrations and (ii) improvement of soil health.
The analysis of the soil collected from the industrial site revealed that the soil was contaminated with 8500 mg kg−1
DW (dry weight) soil of total petroleum hydrocarbons (TPHs) and 5200 mg kg−1
DW of PAHs. Concentration values for the individual PAHs and hydrocarbon fractions were (mg kg−1
DW soil): naphthalene = 120; acenaphthene = 78; fluorine = 82; phenanthrene = 500; anthracene = 140; fluoranthene = 830; pyrene = 570; benzo(a)anthracene = 550; chrysene = 520; benzo(b)fluoranthene = 570; benzo(k)fluoranthene = 250; benzo(a)pyrene = 400; dibenzo(a.h)anthracene = 89; benzo(ghi)perylene = 220; indeno(1.2.3-cd)pyrene = 240; sum of 10 PAH-VROM = 3800; C10-C12 hydrocarbon fraction = 100; C12-C16 hydrocarbon fraction = 380; C16-C21 hydrocarbon fraction = 2400; and C21-C40 hydrocarbon fraction = 5600. At the end of the experiment, prior to their interpretation, soil contaminant concentrations were corrected to take into account the “dilution factor” resulting from the application of the organic amendments. Contaminant concentrations at the end of the experiment are shown in Table 2
. Statistically significant differences were detected for the “Plant x Bioaugmentation” interaction: values of TPHs were significantly lower in bioaugmented planted pots than in non-bioaugmented planted pots; by contrast, values of total-PAHs were higher in bioaugmented planted pots than in non-bioaugmented planted pots. Similarly, statistically significant differences in contaminant concentrations were detected for the “Plant x Amendment” interaction: in planted pots, the addition of dried cow slurry resulted in higher values of total-PAHs with respect to (i) pots amended with composted horse manure and (i) unamended controls (and also with respect to unplanted pots amended with dried cow slurry). In any case, it was concluded that the applied treatments failed at reducing the concentrations of the target soil contaminants (TPHs and PAHs) since, at the end of the experiment, there were no significant differences among treatments, including the untreated control (unplanted, non-bioaugmented control). Then, the observed reduction in contaminant concentrations, compared to the initial values (see above), was most likely due to soil manipulation, not to the applied remediation treatments.
On the other hand, significantly higher values of plant DW were observed in pots amended with dried cow slurry, compared to pots amended with composted horse manure or unamended controls (Figure 1
). Values of total N and CWS
were higher in soils amended with composted horse manure (0.39 ± 0.01% and 636 ± 46 mg C kg−1
soil) and dried cow slurry (0.37 ± 0.03% and 565 ± 65 mg C kg−1
soil) than in control unamended soils (0.28 ± 0.02% and 317 ± 28 mg C kg−1
soil) (Table 3
). Values of soil pH were slightly, not relevantly, higher in soils amended with composted horse manure, whereas a slight increase in electrical conductivity (EC) was observed in soils amended with dried cow slurry (1.7 ± 0.1 dS m−1
) with respect to control soils (1.4 ± 0.1 dS m−1
< 0.06) (Table 3
The application of organic amendments, especially dried cow slurry, increased the values of most of the microbial parameters determined here. Microbial activity parameters (see below) were significantly higher in soils amended with dried cow slurry, followed by soils amended with composted horse manure and, finally, unamended controls (Figure 2
; F = 16.1, p
< 0.002) (Table 4
). According to the variation partitioning analysis, “amendment” application accounted for 64% of the explained variation, while “bioaugmentation” and “plant” growth explained only 1.3 and 2.2% of the variation, respectively. Significantly highest values of soil respiration (4 ± 0.3 mg C kg−1
soil) were, in general, obtained in soils amended with dried cow slurry (Table 4
). Lowest values of soil respiration (1.57 ± 0.3 mg C kg−1
soil) were detected in unamended controls [planted pots showed significantly higher values (1.85 ± 0.07 mg C kg−1
soil) than unplanted pots (1.30 ± 0.01 mg C kg−1
soil)] (Table 4
). Bioaugmentation led to lower values of soil respiration in planted pots amended with composted horse manure and dried cow slurry, compared to unamended controls. Regarding NPM
, values were higher (106 ± 24 mg N-NH4+
DW soil) in soils amended with dried cow slurry (lowest values—6 ± 3 mg N-NH4+
DW soil - were obtained in soils amended with composted horse manure). NPM
values were higher in planted vs. unplanted soils (120 ± 13 and 90 ± 25 mg N-NH4+
DW soil, respectively). Bioaugmentation did not cause significant differences among treatments in regard to NPM
. Concerning soil enzyme activities, the “amendment” factor was significant for all soils, with highest values being detected in soils amended with dried cow slurry. The “plant” factor was significant for 6 enzyme activities (out of 9), with generally higher values observed in planted vs. unplanted pots. No clear effect of the “bioaugmentation” factor was observed for soil enzyme activities.
Regarding parameters that provide information on soil microbial biomass, according to the RDA, values increased in amended soils (Figure 3
). The first axis explained 65% of the variance (F = 21.7; p
< 0.002). The variation partitioning analysis showed that the “amendment” factor was the most important: it explained 58% of the variation, while “plant” and “bioaugmentation” factors explained only 12 and 5% of the variation, respectively. Unexpectedly, in unamended controls, CMB
values were higher in unplanted (1.652 ± 342 mg C kg−1
DW soil) than in planted (977 ± 24 mg C kg−1
DW soil) soils (Table 5
). In general, the bioaugmentation treatment resulted in higher CMB
values. Both the 16S rRNA (total bacteria) and 18S rRNA (total fungi) gene copy numbers increased in amended soils with respect to controls. The highest gene copy numbers were found in bioaugmented planted soils amended with dried cow slurry: 3.9×108
for 16S rRNA and 30e 5
for 18S rRNA.
Regarding microbial diversity parameters, the duration of the lag phase and t1/2
values (time corresponding to the middle of the exponential phase), calculated from the growth curves of the BiologTM
CLPPs, were significantly shorter in soils amended with dried cow slurry (Table 6
). In amended controls and soils amended with composted horse manure, planted pots showed significantly lower values of lag phase and t1/2
, compared to unplanted pots. The highest slopes of the CLPPs growth curves were observed in soils amended with dried cow slurry (Table 6
). In unplanted soils, AWCDt1/2
values were lower in bioaugmented vs. non-bioaugmented pots. Finally, values of NUSt1/2
and Shannon’s diversity were significantly lower in soils amended with dried cow slurry, compared to unamended controls. According to the RDA performed with the slope values of all the substrate utilization profiles, values were higher in planted soils and soils amended with dried cow slurry (Figure 4
). The variation partitioning revealed that the “plant” and “amendment” factors explained 19 and 15% of the variability, respectively.
As far as structural microbial diversity is concerned, our metabarcoding data revealed no significant differences among treatments in terms of Shannon’s diversity. On the contrary, Simpson’s diversity was lower in soils amended with composted horse manure (0.9898), compared to unamended controls (0.9922) and soils amended with dried cow slurry (0.9929) (Table S2
). Pielou’s evenness was higher in soils amended with dried cow slurry (0.76), compared to unamended controls (0.74) and soils amended with composted horse manure (0.74). Rarefied richness was significantly lower in soils amended with composted horse manure (4080), compared to unamended controls (3770) and soils amended with dried cow slurry (3750). The clustering analysis (Figure 5
) clearly separated unamended controls, soils amended with composted horse manure, and soils amended with dried cow slurry. These results were also reflected in the distribution of the most abundant families (Figure 6
). The most abundant families were Cytophagaceae
(especially in soils amended with composted horse manure), Xanthomonadaceae
(in soils amended with dried cow slurry), Rhodospirillaceae
(in both unamended controls and soils amended with dried cow slurry). Regarding the bacterial strains used in our bioaugmentation consortium, Pseudomonadaceae
doubled its abundance in bioaugmented vs. non-bioaugmented soils. This increase was not observed for Bukholderia
All the applied treatments failed at achieving a reduction in the concentration of the target contaminants (TPHs, PAHs), but some (especially, biostimulation with organic amendments) did succeed at improving soil health. Particularly, the application of dried cow slurry enhanced soil health. In consequence, and taking into consideration an often-mentioned remediation paradigm which states that “the goal of any remediation treatment must be not only to reduce the concentration of the target contaminants but, most importantly, to also improve soil health”, our results point out a not-so-uncommon situation in which remediation efforts fail from the point of view of the reduction in contaminant concentrations while succeeding to improve soil health. Then, it cannot be concluded that our remediation attempts were completely successful (we achieved no significant decrease in contaminant concentrations) but, in a sense, they were partly successful because they resulted in an improved health of the long-term contaminated industrial soil.
This abovementioned situation is the opposite of that often encountered when applying many physicochemical methods of soil remediation, i.e., a reduction in contaminant concentrations is achieved at the expense of negatively affecting soil health. Sadly, despite the abovementioned paradigm, we are used to “remediating” a contaminated site using techniques that strongly (at times, irreversibly) alter the functionality of the treated soil, and claim to have been successful if contaminant concentrations have been reduced below regulatory limits. It is evident that when soil contaminants are causing negative effects on human health, we must remove those contaminants whatever the costs for the soil ecosystem (though, preferably, with the minimum harm to the soil ecosystem and the environment in general). But, in many cases, due to their lack of solubility, mobility, bioavailability, bioaccessibility, etc., or to the lack of a relevant exposure route from the contaminants to humans, the damage caused by some remediation methods on the integrity of the soil ecosystem might be more detrimental than the actual harm caused by the contaminants themselves. In this respect, biological remediation methods are usually less damaging, if at all, to the integrity of the soil ecosystem.
Finally, to achieve a significant reduction in the concentration of the target contaminants in the studied industrial soil (i.e., the most important objective from an anthropocentric and, in particular, legal point of view) using the same biological remediation strategies tested here, much research is particularly needed on (i) the nature and dose of application of the surfactants required to increase the bioavailability and degradability of recalcitrant organic contaminants; and (ii) the ecological fitness of the bacterial strains used for bioaugmentation (including competitive traits such as growth rate, mobility, capacity to express the specific degradation genes under those conditions, etc.).