**2. Methods used to analyze the CO2 microbial production in soil**

#### **2.1 CO2 sampling**

70 Management of Organic Waste

was used to designate the hydrocarbon tails of crude oil introduced into soil and transformed into the product that lost the original properties (i.e., the quantitative ratio of hydrocarbon components changed and the organic products of microbial biosynthesis appeared, which differ from the initial oil components in metabolic availability for a wide range of soil microorganisms, etc). It has been known that soil microbial communities are able to adjust to unfavourable conditions and to use a broad spectrum of substrates (Jobson et al. 1974; Nikitina et al. 2003). They have unique metabolic systems that allow them to utilise both natural and anthropogenic substances as a source of energy and tissue constituents. These unique characteristics make the microbiota useful tool in monitoring and remediation processes. Bioremediation of soil contaminated with oil hydrocarbons has been established as an efficient, economic, versatile, and environmentally sound treatment (van Hamme et al. 2003). Several reports have already focused on the composition of natural microbial populations contributing to biotransformation and biodegradation processes in different environments polluted with hydrocarbons (Juck et al. 2000; Hamamura et al. 2008; Marques et al. 2008). It is becoming increasingly evident that the fate of anthropogenic hydrocarbons pollutants entering the soil system requires efficient monitoring and control. The bioremediation potential of microbial communities in soil polluted with oil hydrocarbons depends on their ability to adapt to new environmental conditions (Mishra et al. 2001; Kaplan and Kitts 2004). Investigations into how bioremediation influences the response of a soil microbial community, in terms of activity and diversity, are presented in a series of publications (Jobson et al. 1974; Margesin and Schinner 2001; Zucchi et al. 2003; Hamamura et al. 2006; Margesin et al. 2007). The methods of monitoring and characterization of hydrocarbon degrading activity of soil microbiota are of special interest (Margesin and Schinner 2005; Abbassi and Shquirat 2008; Pleshakova et al. 2008). Oil hydrocarbon biodegradation and transformation in soils can be monitored by estimating the concentration of pollutant (Tzing et al. 2003) and the formation of respective metabolites. The most ubiquitous and universal metabolites is carbon dioxide (CO2), since respiration is

by far the prominent pathway of biologically processed carbon.

The activity of soil microbiota can be characterized by the method of the substrate-induced respiration (SIR) which was used for the measurement of CO2 production and the estimation of soil microbial biomass. When an easily microbial degradable substrate, such as glucose, is added to a soil, an immediate increase of the respiration rate is obtained, the size of which is assumed to be proportional to size of the microbial biomass (Anderson and Domsch 1978). In addition to SIR, the index of the specific microbial activity in soil is the priming effect (PE) of introduced exogenous substrate, which was defined as 'the extra decomposition of native soil organic matter in a soil receiving an organic amendment" (Bingeman et al. 1953). The PE may be represented by the following three indices: (a) positive PE shows that exogenous substrate introduction concurrent with its mineralization increases SOM mineralization to a rate exceeding the previous rate; (b) zero PE shows that CO2 is produced additionally only as a result of microbial mineralization of introduced substrate without changing the existing rate of SOM mineralization; and (c) negative PE values show that exogenous substrate introduction decreases SOM mineralization rate and CO2 production is determined mainly by mineralization of the substrate. PE determination only by the difference of CO2 production rate before and after substrate introduction into soil suffers from the known uncertainly of CO2 sources and does not allow distinguishing between the so-called "real" and "apparent" PE. (Blagodatskaya et al. 2007; Blagodatskaya Soil samples, 100 g dry weight, were placed into 700-ml glass vials, hermetically closed and pre-incubated for 3 days at 22 0С. Metabolic carbon dioxide (CO2) formed by microbial mineralization of SOM and test-substrate (crude oil) was collected using glass plates (10 ml) placed the over soil surface, containing 2-3 ml of 1M NaOH solution. Production of СО2 in the course of the experiment in each of the vials was determined by titration of the residual alkali in the plates using an aqueous 0.1M HCl solution. The total amount of СО2 fixed in the NaOH solution was also determined by precipitation with BaCl2 and quantitative retrieval of BaCO3. Barium carbonate was washed with water, precipitated, dried, and the resulting precipitate weighed and used for quantitative calculation of metabolic СО<sup>2</sup> production and carbon isotope analysis.

### **2.2 The kinetics of CO2 respiration**

Specific CO2 evolution rates (µ) of soil microorganisms after crude oil addition to soil were estimated from the kinetic analysis of substrate-induced respiration (*CO2(t*)) by fitting the parameters of equation [1]:

$$\text{CO2}(\text{t}) = \text{K} + \text{r } \exp(\mu \text{ t}) \tag{1}$$

where *K* is the initial respiration rate uncoupled from ATP production, *r* is the initial rate of respiration by the growing fraction of the soil microbiota which total respiration coupled with ATP generation and cell growth, and *t* is time (Panikov and Sizova 1996; Stenström et al. 1998; Blagodatsky et al. 2000). The lag period duration (*tlag*) was determined as the time interval between substrate addition and the moment when the increasing rate of microbial growth-related respiration *r·exp(µ·t)* became as high as the rate of respiration uncoupled from ATP generation.

$$\mathfrak{th}\_{\log} = \text{Im}(\mathbf{K}/\mathbf{r})/\mu \tag{2}$$

According to the theory of microbial growth kinetic (Panikov 1995; Blagodatskaya et al. 2009), the lag period was calculated by using the parameters of approximated curve of respiration rate of microorganisms with [2].

#### **2.3 Carbon isotopic analysis**

The metabolic activity of soil microbial community with respect to substrate (crude oil hydrocarbons) was determined from CO2 evolution rates and the 13C-CO2 isotope signature. The characteristics of abundance ratios of carbon isotopes 13C/12C in SOM, crude oil, and metabolic СО2 (as BaCO3) were measured using by isotopic mass-spectrometry (Breath MAT-Thermo Finnigan) connected with a gas chromatograph via ConFlow interface. Isotope analysis of metabolic СО2 was performed using about 3-4 mg of obtained BaCO3 [M = 197.34], which then was degraded to СО2 by orthophosphoric acid in a 10-ml container. For the analysis of carbon isotope contents of organic matter, SOM and crude oil samples were combusted to СО2 in ampoules at 560 0С in the presence of copper oxide.

The ratios of peak intensities in СО2 mass spectra with m/z 45 (13C16O2) and 44 (12C16O2) were used for quantitative characterization of the content of 13C and 12C isotopes in the analyzed samples. According to the accepted expression [3], the amount of 13C isotope was determined in relative units 13C (‰):

$$\text{\\$}^{13}\text{C}^{\circ} = \text{\{R}\_{\text{sa}} / \text{R}\_{\text{st}} \text{--1\} 1000 \text{ \%o}}\tag{3}$$

where Rsa=(13C)/(12C) represented the abundance ratios of isotopes 13C /12C in a sample and Rst=(13C)/(12C) was the ratio of these isotopes in the International Standard PDB (Pee Dee Belemnite) (Craig 1957). Each СО2 sample was analyzed in three repeats; standard error was about 0.1‰. The 13C values are characteristics of stable isotope composition or the 13C/12C abundance ratio in the analyzed compounds. Negative values indicate the 13C depletion; positive values indicate 13C enrichment relative to PDB standard.

#### **2.4 Mass isotope balance**

Metabolic carbon dioxide produced in the experiments and controls was accumulated during the appropriate time intervals (1-3 days) followed by determination of its quantity and carbon isotope characteristics. The average weighed carbon isotope composition of metabolic СО2 (13Cave), which was obtained in detached time intervals, was determined using the expression [4]:

$$\text{"\\$}^{13}\text{C}\_{\text{ave}} = \left(\sum \text{q}\_{\text{i}} \text{ @}^{13}\text{C}\_{\text{i}}\right) / \sum \text{q}\_{\text{i}} \text{ @} \tag{4}$$

where qi and 13Ci were СО2 production rate and carbon isotope composition at *i*–intervals, respectively.

Determination of mass isotope balance is based on the suggestion that the characteristics of carbon isotope content (δ13C) of CO2 produced during microbial mineralization of hydrocarbons will inherit the δ13C value of crude oil with an accuracy of isotopic fractionation effect. According to (Zyakun et al. 2003), the δ13C value of metabolic CO2

equation [5]:

as [9]:

[10], where *i* varied from 1 to *n*:

samples, respectively.

are presented, by definition, as [6] and [7]:

The Waste Oil Resulting from Crude Oil Microbial Biodegradation in Soil 73

produced during oxidation of n-hexadecane and aliphatic hydrocarbons was less by 1-3 ‰ compared to the isotope characteristics of substrates used. It means that the δ13C value of CO2 produced during microbial degradation of oil hydrocarbons was estimated by δ13C equal to the value over a rang of -28 to -31 ‰, where δ13C of the crude oil was about of δ13Coil = –28,40,2 %o. It is rather different from CO2 resulting from soil organic matter (SOM) mineralization (δ13CSOM is equal to -23,5±0,5 ‰ for the soil). Thus, after addition of the oil hydrocarbon to soil, the mass isotope balance for CO2 evolved during microbial mineralization of SOM and the exogenous substrate (SUB) was calculated using

where δ13CSOM and δ13CMIX are isotopic characteristics of 13C content in CO2 before and after substrate addition to the soil; δ13CSUB is the isotopic characteristic of 13C content in CO2 produced during microbial mineralization of the test substrate; QSOM and QSUB are CO2 quantities resulted from microbial mineralization of SOM and added substrate in the soil

Here the shares of СО2 formed by mineralization of SOM (FSOM) and oil hydrocarbons (FSUB)

FSOM= QSOM /(QSOM + QSUB) (6)

 FSUB=(1-FSOM)= QSUB/(QSOM + QSUB) (7) Using carbon isotope characteristics of total СО2 formed by microbial mineralization of SOM and oil hydrocarbons (13Ctot) (in experiments) and СО2 formed by mineralization of only SOM (13CSOM) (in controls) and assuming that СО2 produced by oil mineralization inherits its isotope composition (13Coil), respectively, the share of СО2 formed by

FSOM = (13Ctot - 13Coil)/(13CSOM - 13Coil) (8)

Cumulative CO2 produced during the microbial substrate oxidation was calculated as follows. The ΔQi quantity of CO2 evolved during the Δti-time interval (i = 1,2, …,n) was estimated as ΔQi = Δti·vi, where the vi-value is the rate of CO2 evolved during the time interval Δti. Using δ13Csoil, δ13CSubst and δ13CCO2(mix)(i), the fraction of CO2 resulting from the exogenous substrate (crude oil hydrocarbons) oxidation during Δti can be calculated

where FSOM(i) value can be estimated using equation [8]. The cumulative CO2 quantity (QSubst(CO2)) resulting from microbial oxidation of the substrates in soils was presented by

QSubst(CO2) =Σ ΔQSubst(i) (10)

ΔQSubst(i)=(1-FSOM(i))·ΔQi (9)

mineralization of SOM (FSOM) in experiments was calculated by expression [8].

**2.5 Cumulative CO2 resulted from hydrocarbon mineralization** 

δ13CSOM×QSOM + δ13CSUB×QSUB = δ13CMIX×(QSOM + QSUB) (5)

 tlag=ln(K/r)/µ (2) According to the theory of microbial growth kinetic (Panikov 1995; Blagodatskaya et al. 2009), the lag period was calculated by using the parameters of approximated curve of

The metabolic activity of soil microbial community with respect to substrate (crude oil hydrocarbons) was determined from CO2 evolution rates and the 13C-CO2 isotope signature. The characteristics of abundance ratios of carbon isotopes 13C/12C in SOM, crude oil, and metabolic СО2 (as BaCO3) were measured using by isotopic mass-spectrometry (Breath MAT-Thermo Finnigan) connected with a gas chromatograph via ConFlow interface. Isotope analysis of metabolic СО2 was performed using about 3-4 mg of obtained BaCO3 [M = 197.34], which then was degraded to СО2 by orthophosphoric acid in a 10-ml container. For the analysis of carbon isotope contents of organic matter, SOM and crude oil samples

The ratios of peak intensities in СО2 mass spectra with m/z 45 (13C16O2) and 44 (12C16O2) were used for quantitative characterization of the content of 13C and 12C isotopes in the analyzed samples. According to the accepted expression [3], the amount of 13C isotope was

where Rsa=(13C)/(12C) represented the abundance ratios of isotopes 13C /12C in a sample and Rst=(13C)/(12C) was the ratio of these isotopes in the International Standard PDB (Pee Dee Belemnite) (Craig 1957). Each СО2 sample was analyzed in three repeats; standard error was about 0.1‰. The 13C values are characteristics of stable isotope composition or the 13C/12C abundance ratio in the analyzed compounds. Negative values indicate the 13C

Metabolic carbon dioxide produced in the experiments and controls was accumulated during the appropriate time intervals (1-3 days) followed by determination of its quantity and carbon isotope characteristics. The average weighed carbon isotope composition of metabolic СО2 (13Cave), which was obtained in detached time intervals, was determined

where qi and 13Ci were СО2 production rate and carbon isotope composition at *i*–intervals,

Determination of mass isotope balance is based on the suggestion that the characteristics of carbon isotope content (δ13C) of CO2 produced during microbial mineralization of hydrocarbons will inherit the δ13C value of crude oil with an accuracy of isotopic fractionation effect. According to (Zyakun et al. 2003), the δ13C value of metabolic CO2

13C = (Rsa/Rst –1) 1000 ‰ (3)

13Cave= (∑qi,·13Ci )/∑qi, ‰ (4)

were combusted to СО2 in ampoules at 560 0С in the presence of copper oxide.

depletion; positive values indicate 13C enrichment relative to PDB standard.

respiration rate of microorganisms with [2].

**2.3 Carbon isotopic analysis** 

determined in relative units 13C (‰):

**2.4 Mass isotope balance** 

using the expression [4]:

respectively.

produced during oxidation of n-hexadecane and aliphatic hydrocarbons was less by 1-3 ‰ compared to the isotope characteristics of substrates used. It means that the δ13C value of CO2 produced during microbial degradation of oil hydrocarbons was estimated by δ13C equal to the value over a rang of -28 to -31 ‰, where δ13C of the crude oil was about of δ13Coil = –28,40,2 %o. It is rather different from CO2 resulting from soil organic matter (SOM) mineralization (δ13CSOM is equal to -23,5±0,5 ‰ for the soil). Thus, after addition of the oil hydrocarbon to soil, the mass isotope balance for CO2 evolved during microbial mineralization of SOM and the exogenous substrate (SUB) was calculated using equation [5]:

$$\rm O^{13}\rm C\_{\rm SOM} \times Q\_{\rm SOM} + \rm O^{13}\rm C\_{\rm SUB} \times Q\_{\rm SUB} = \rm O^{13}\rm C\_{\rm MIN} \times (Q\_{\rm SOM} + Q\_{\rm SUB}) \tag{5}$$

where δ13CSOM and δ13CMIX are isotopic characteristics of 13C content in CO2 before and after substrate addition to the soil; δ13CSUB is the isotopic characteristic of 13C content in CO2 produced during microbial mineralization of the test substrate; QSOM and QSUB are CO2 quantities resulted from microbial mineralization of SOM and added substrate in the soil samples, respectively.

Here the shares of СО2 formed by mineralization of SOM (FSOM) and oil hydrocarbons (FSUB) are presented, by definition, as [6] and [7]:

$$\mathbf{F}\_{\text{SCM}} = \mathbf{Q}\_{\text{SCM}} \Big/ \left( \mathbf{Q}\_{\text{SCM}} + \mathbf{Q}\_{\text{SVB}} \right) \tag{6}$$

$$\mathbf{F\_{SUB}} = \begin{pmatrix} 1 \text{-} F\_{\text{SOM}} \end{pmatrix} = \mathbf{Q\_{SUB}} \left/ \left( \mathbf{Q\_{COM}} + \mathbf{Q\_{SUB}} \right) \right. \tag{7}$$

Using carbon isotope characteristics of total СО2 formed by microbial mineralization of SOM and oil hydrocarbons (13Ctot) (in experiments) and СО2 formed by mineralization of only SOM (13CSOM) (in controls) and assuming that СО2 produced by oil mineralization inherits its isotope composition (13Coil), respectively, the share of СО2 formed by mineralization of SOM (FSOM) in experiments was calculated by expression [8].

$$F\_{\rm{SOM}} = \left( \delta \text{rc} \mathbf{C}\_{\rm{tot}} \text{ - } \delta \text{rc} \mathbf{C}\_{\rm{oil}} \right) / \left( \delta \text{rc} \mathbf{C}\_{\rm{SOM}} \text{ - } \delta \text{rc} \mathbf{C}\_{\rm{oil}} \right) \tag{8}$$

#### **2.5 Cumulative CO2 resulted from hydrocarbon mineralization**

Cumulative CO2 produced during the microbial substrate oxidation was calculated as follows. The ΔQi quantity of CO2 evolved during the Δti-time interval (i = 1,2, …,n) was estimated as ΔQi = Δti·vi, where the vi-value is the rate of CO2 evolved during the time interval Δti. Using δ13Csoil, δ13CSubst and δ13CCO2(mix)(i), the fraction of CO2 resulting from the exogenous substrate (crude oil hydrocarbons) oxidation during Δti can be calculated as [9]:

$$
\Delta \mathbf{Q}\_{\text{Subst(i)}} = (\mathbf{1} \cdot \mathbf{F}\_{\text{SOM(i)}}) \cdot \Delta \mathbf{Q}\_{\text{i}} \tag{9}
$$

where FSOM(i) value can be estimated using equation [8]. The cumulative CO2 quantity (QSubst(CO2)) resulting from microbial oxidation of the substrates in soils was presented by [10], where *i* varied from 1 to *n*:

$$\mathbf{Q}\_{\text{Subbt}(\text{CO2})} = \Sigma\_{\text{-}} \Delta \mathbf{Q}\_{\text{Subt}(\text{i})} \tag{10}$$

The Waste Oil Resulting from Crude Oil Microbial Biodegradation in Soil 75

fractions having values -28.9 %o and -27.2 %o, respectively. The isotopic characteristics (13C) of the oil used in the experiments were found to be close to the samples of crude oil from oilfields of the Arabian region, where the 13C value was –27.5 0.5 ‰ for oil, -28 0.5 ‰ for alkane fraction, and -26.5 1.5 ‰ for the fraction containing mainly aromatic

To estimate the potential of microbial mineralization of oil hydrocarbons polluted soils, the CO2 production was determined in 12 glass vials with tested soils (three replicates of each experiment and control) (Table 1). In Experiment 1, crude oil was introduced into vials with native soil containing only native soil microorganisms; in Experiment 2, the laboratory strain *Pseudomonas aureofaciens* BS1393(pBS216) (Kochetkov et al. 1997) was additionally introduced into the same soil with oil. Native soil without oil and the same soil with the

The strain *Pseudomonas aureofaciens* BS1393(pBS216) bears the plasmid pBS216 that controls naphthalene and salicylate biodegradation, is able to utilize aromatic oil hydrocarbons, and has an antagonistic effect on a wide range of phytopathogenic fungi (Kochetkov et al. 1997). The ability of the strain to synthesize phenazine antibiotics and thus staining its colonies bright-orange on LB agar medium allowed its use as a marker of quantitative presence of the above microorganisms in soil in the presence of aboriginal microflora ( Sambrook, et al.

The introduced strain was previously grown in liquid LB medium till stationary phase (28°С, 18 h) and then uniformly introduced into soil to a concentration of 106 cells g-1 soil. The control of the bacteria strain growth was accomplished weekly during 67 days. A composite soil sample was collected from three separate sub-samples from the vial and analyzed for bacterial quantities. Approximately one g of the composite soil sample was suspended in 10 ml of 0.85% NaCl on "Vortex", soil particles were precipitated, and 1 ml of supernatant was used for making dilutions (10×-10000×). Volume of 0.1 ml of the

*Control* 2: Native soil with soil microbiota + *Pseudomonas aureofaciens* BS1393(pBS216) (three of glass vials)

*Experiment* 2: Native soil with soil microbiota + crude oil+ *Pseudomonas aureofaciens* BS1393(pBS216) (three of glass vials)

strain BS1393(pBS216) were used as controls 1 and 2, respectively (Table 1).

hydrocarbons, respectively (Belhaj et al. 2002).

*Control* 1: Native soil with soil microbiota (three of glass vials)

*Experiment* 1: Native soil with soil microbiota + crude oil (three of glass vials)

Table 1. Scheme of experiments and controls

**3.3 Microorganisms** 

1989].
