**4. Key study: rhizobiota volatilome changes under challenges of pharmaceuticals**

#### **4.1 Experimental setup**

Based on the frequency of their presence in the environment, the following nonsteroidal anti-inflammatory drugs were choosing for experiment: ibuprofen, ketoprofen, and diclofenac. Commonly consumed aromatic plants such as sage (*Salvia officinalis* L.), dill (*Anethum graveolens*), and rosemary (*Rosmarinus officinalis* L.) were used in the experiment. Argic phaeozem soil, free of studied pharmaceuticals, was used in this experiment. Soil material was contaminated individually with each of these pharmaceuticals with the following theoretical concentrations: 0.7 mg⋅kg−1 diclofenac, 0.5 mg⋅kg−1 ibuprofen, and 0.2 mg⋅kg−1 ketoprofen. Ten-day seeds of the selected plant materials were planted in pots containing approximately 1.2 kg of contaminated soils and allowed for development until maturity. Plant growth was performed in laboratory in a climate chamber with the following conditions: day – 24°C, 12 h of light; night – 18°C, 12 h; soil water-holding capacity was adjusted to 58% during the experiment. Control samples without soil contamination were grown in similar conditions. Each experiment was performed with three pots in parallel. Rhizosphere soil samples were collected after each plant has reached maturity. The schematic presentation of experiment setup is shown in **Figure 1**. From each rhizosphere 1 g of soil was collected for soil microbiota community assessment and 1 g of soil for microbial volatile organic compounds analysis.

#### *Biodegradation Technology of Organic and Inorganic Pollutants*

**Figure 1.** *Experiment setup.*

#### **4.2 Gas chromatographic assessment of rhizosphere microbiota community**

One gram of lyophilized rhizosphere soil was used for microbiota phenotypic structure and abundance analysis. Assessment was performed applying phospholipids-derived fatty acids (PLFA) gas chromatographic approach. Phospholipidsderived fatty acids were extracted according to the method presented by Blight and Dyer [32], and Frostegard et al. [33] and derivatized for gas chromatographic analysis (7890A GC-FID, Agilent Technologies, Santa Clara, CA, USA). Their detection was done with flame ionization detector. Separation of all fatty acid methyl esters from each extract was done with a 5% phenyl-methyl polysiloxane column (25 mm × 0.2 mm id., 0.33 μm film thickness, HP-Ultra 2, J&W Scientific, Folsom, CA, USA). Helium was used as carrier with 1.2 mL·min−1 flow. Detector and injector temperature was set at 300 and 280°C, respectively. Oven temperature program starts at 170°C followed by an increase with 28°C·min−1 until 288°C, continued with an increase with 60°C·min−1 until 310°C. This final temperature was maintained isotherm for 1.25 min. Phenotypic profile of rhizobiota based on PLFA profile was determined using the MIDI SherlockTM Microbial Identification System software (Microbial ID, Inc., Newark, DE, USA). Also, the following PLFA biomarkers were used to identify saprotrophic fungi, ectomycorrhizal fungi, nitrogen-reducing bacteria and sulfur-reducing bacteria: 18: 2ω6c – saprotrophic fungi; 18:2ω9c – ectomycorrhizal fungi; 18:2ω6c and 18:3ω3 – nitrogen-reducing bacteria; and 17:1ω7c, 10Me16:0, 17:1ω6, 15:1, i17:1ω7c, cy18:0ω7.8, i15:1ω7c and i19:1ω7c for sulfur-reducing bacteria [32, 33].

#### **4.3 Gas chromatographic–mass spectrometric in profiling rhizobiota volatilomes**

Volatile organic compounds emitted by rhizobiota were assessed through headspace-solid-phase microextraction sampling using 85 μm polyacrylate fiber (Supelco Inc., Bellefonte, PA, USA). For this analysis, 1 g of rhizosphere soil was diluted with 2 mL of PBS solution in 20 mL headspace glass vials

*Gas Chromatographic: Mass Spectrometric Mining the Volatilomes Associated to Rhizobiota… DOI: http://dx.doi.org/10.5772/intechopen.102895*

(Agilent Technologies). The tightly capped headspace vials were incubated for 72 h in dark at 25°C. After this period, the vials were equilibrated for 30 min at 60°C using a TriPlus RSH autosampler (Thermo Scientific, Austin, TX, USA). Thermally activated SPME fiber was inserted in the vial headspace surface and kept for 15 min to allow the adsorption of volatile organic compounds on the fiber extractive phase. Rhizobiota volatilome analysis was conducted on gas chromatography–mass spectrometry (GC–MS/MS, Trace 1310, TSQ 9000, Thermo Scientific, Austin, TX, USA). Ionization was carried out in electron impact mode at 70 eV ionization energy. Volatile organic compounds were separated on a HP-5MS capillary column 30 m × 0.25 mm, 0.25 μm). The carrier gas was He with 1.2 mL·min−1 flow. The SPME fiber with adsorbed volatile organic compounds was inserted into the GC injection port at 250°C for 5 min to allow the desorption of analytes. The volatile organic compounds were identified by comparison of their mass spectra with compounds corresponding to mass spectra library (NIST/EPA/NIH, Chromeleon 7.2 CDS Software, Thermo Scientific, Austin, TX, USA). All identified volatile organic compounds were expressed in percentages as a normalized amount of each volatile organic compound resulted after the division of peak areas of identified volatile organic compounds by total peak area of all identified volatile organic compounds.

#### **4.4 Rhizobiota differentiation between studied plant species rhizosphere**

Total abundance of control samples of rhizosphere soil of *Rosmarinus officinalis* L., *Anethum graveolens,* and *Salvia officinalis* L. microbiota varies within the range of 216.6–191.8 nmol⋅g−1. Higher abundance was identified in *R. officinalis* L. rhizosphere, followed by *A. graveolens* and *S. officinalis* L. Bacterial dominance was observed in all cases, bacterial PLFA:total PLFA being higher than 0.8. Representative bacterial groups were Gram-negative bacteria, followed by Grampositive and aerobe bacteria group. Gram-negative bacteria abundance represented 82.3% in *R. officinalis* L., while in rhizosphere of *A. graveolens* and *S. officinalis* L. represented 79.1 and 68.5% (see **Figure 2**).

Fungal community represented approximately 14% of the total microbial abundance, with higher abundance in *S. officinalis* L. – 16.2%.

### **4.5 Rhizobiota emitted volatile organic compounds variation studied rhizosphere**

In control rhizosphere of the three aromatic plants, the main emitted volatile organic compounds measured through GC–MS were terpenes, alcohols, aromatic compounds, ketones, and organic acids. Identified volatile organic compounds percentage amount is listed in **Table 1**.

Terpenes were determined in higher amount in all rhizosphere soil with an average amount of 26%. Higher content of terpene compounds was measured in *R. officinalis* L. (30%) followed by *A. graveolens* (28%) and *S. officinalis L*. (20%). In case of *S. officinalis* L. rhizosphere, the second representative group of volatile organic compounds was organic acids (13.4%) followed by alkane compounds (12.6%). In the rhizosphere soil of *A. graveolens* and *R. officinalis* L., the second prevalent group was alkane for both cases. In all rhizosphere soils, ester compounds were found in lower amount (< 5%).

#### **4.6 Pharmaceuticals' influence on rhizobiota community and volatilome profile**

Rhizosphere microbiota total PLFA ranged between 216.6 and 167.6 nmol⋅g−1 dry weight soil. Between studied plant species during all experiment cases,

#### **Figure 2.**

*Bacterial communities' abundance variation in studied rhizosphere soils. a.) Rosmarinus officinalis L.; b.) Anethum graveolens; c.) Salvia officinalis L.*

it was observed that the rhizosphere microbiota abundance was higher in case of *R. officinalis* L. rhizosphere soil followed by *A. graveolens* and *S. officinalis* L. (see **Figure 3**). Rhizobiota community phenotypic profile revealed bacterial dominance, bacterial PLFA:total PLFA >0.837. Gram-negative bacteria were the most representative bacterial group in studied rhizosphere soils. Their abundance was within 56.8–82.3 nmol⋅g−1. They were followed by Gram-positive bacteria and aerobe bacteria. PLFA ratio among aerobe bacteria and anaerobe bacteria was higher than 3.6 for *S. officinalis* L. rhizosphere and 1.4 for *A. graveolens* and *R. officinalis* L., these data clearly evidence aerobic bacteria dominance. Fungal community was represented within 9.5–16.3% of the total rhizobiota abundance in studied experiments.

Influence of studied NSAIDs on rhizosphere microbial community was observed from experimental data. Compared with control samples, in all cases was seen a decrease in total abundance. *Rosmarinus officinalis* L. rhizosphere total abundance decreased with 8.5% under exposure to ketoprofen, followed by a decrease with 7.4% at diclofenac exposure and 5.1% at ibuprofen exposure. Compared with control samples, *Anethum graveolens* rhizosphere microbiota decreased with 13% in case of exposure to diclofenac and ketoprofen and with 11% in case of exposure to ibuprofen. *Salvia officinalis* L. rhizosphere microbial community did not decrease when it was exposed at diclofenac but presented a lower abundance with 12.6% and 5.6% when it was exposed to ibuprofen and ketoprofen, respectively.


*Gas Chromatographic: Mass Spectrometric Mining the Volatilomes Associated to Rhizobiota… DOI: http://dx.doi.org/10.5772/intechopen.102895*


#### **Table 1.**

*Percentage value of emitted volatiles in control samples rhizosphere.*

#### **Figure 3.**

*Total microbial, bacterial, and fungal abundance variation in studied aromatic plants rhizosphere exposed at different NSAIDs.*

Principal component analysis revealed that rhizosphere microbial community structure could change under exposure to specific NSAIDs (**Figure 4**). Grampositive bacteria, fungi, and methanotroph bacteria in *S. officinalis* L. rhizosphere

*Gas Chromatographic: Mass Spectrometric Mining the Volatilomes Associated to Rhizobiota… DOI: http://dx.doi.org/10.5772/intechopen.102895*

#### **Figure 4.**

*Principal component analysis of NSAIDs' impact on rhizosphere soils microbial communities. a.) Salvia officinalis L.; b.) Rosmarinus officinalis L.; c.) Anethum graveolens.*

presented a positive correlation in the presence of diclofenac while Gram-negative bacteria and ectomycorrhizal fungi decreased in the presence of ibuprofen (**Figure 4a**). This was explained by principal component analysis in 77.3%. Ibuprofen presence has a negative impact on methanotroph bacteria, N-reducing bacteria, and saprotrophic fungi in *R. officinalis* L. rhizosphere. Diclofenac's presence did not show significant impact on Gram-positive and aerobe bacteria (PC1 58%, PC2 25%; **Figure 4b**). Diclofenac's presence was also negatively correlated with N-reducing bacteria and Gram-positive bacteria abundance in *A. graveolens* rhizosphere (**Figure 4c**).

Emitted volatile organic compounds in studied rhizosphere changed over exposure to NSAIDs. In case of *S. officinalis* L. rhizosphere, the higher decrease was observed for alcohol compounds and organic acids in the presence of ibuprofen (< 15%), especially in case of hexan-1-ol, butane-2.3-diol and propanoic acid. Under contamination with diclofenac, aromatic compounds increased slightly (> 2%). Rhizosphere emitted volatile organic compounds of *A. graveolens* presented an increase with approximately 5–10% in case of phenol, germacrene, methyl eugenol, butanoic acid, and nonanal in the presence of ketoprofen while indole decreased with approximately 30% in case of exposure to ibuprofen and diclofenac. *R. officinalis* L. rhizosphere emitted volatile organic compounds changed significantly in the presence of ibuprofen and diclofenac. The following decrease was observed compared with control samples: terpene content <12%, organic acids <7% and alkane <4%.
