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

Anatoly M. Zyakun, Vladimir V. Kochetkov and Alexander M. Boronin *Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS Russia* 

#### **1. Introduction**

68 Management of Organic Waste

Valocká, B., Dubinský, P., Papajová, I., Sabová, M. (2000). Effect of anaerobically digested

conditions. Helminthologia, 37, p. 53-57.

pig slurry from lagoon on soil and plant nematode communities in experimental

Environmental pollution by oil and oil products, which occurs at petroleum extraction wells, as a result of spills from oil tankers, pipe line breaks, disposal of refinery waste, leaks at gasoline stations, etc., have caused tremendous damage to ecological systems especially to many plant species (Adam and Duncan 2002; 2003; Palmroth et al. 2005), and a wide array of animals (Khan and Ryan 1991; Tevvors and Sair 2010). According to available data (Wang et al. 2011), the total amount of all major spills in the world was about 37 billion barrels of crude oil pollute soil and water ecosystems. It exceeds the total amount of crude oil consumption for the entire world annually (30 billion barrels in 2006) (Mundi 2010). Consequently, the problem of environmental pollution with anthropogenic hydrocarbons and their influence on natural ecosystems calls for comprehensive investigation. Crude oil consists of a number of rather complicated components, which are toxic and can exert side effects on environmental systems. Oil pool contains aliphatic and polycyclic aromatic hydrocarbons, for example, crude oil consists of alkanes 15 - 60 %, naphthenes 30-60 %, aromatics 3-30% and asphaltenes 6 % by weight ( Speight 1990 ). The extent of oil spills can have a legacy for decades, evens centuries in future (Wang et al. 2011). Toxic effects of oil and oil products on the soil environment include increasing hydrophobicity of soils and disruption of water availability to vegetation, and direct toxicity to plants and microorganisms. At the sub-toxic level, negative effects may include the absorption of lowmolecular oil hydrocarbons into plant tissues, and the inhibition or activation of microbial soil processes. The soil, although is an important sink for a wide range of substances, pollutant load exceeding certain threshold has the potential of impacting negatively on the capacity of the soil to perform its ecosystem functions with repercussions on sustainability issues such as plant growth and some non-hydrocarbon utilizing microorganisms. For instance, the aromatics in crude oil produce particular adverse effect to the local soil microbiota. It was found that phenolic and quinonic naphthalene derivatives inhibited the growth of some microbial cells (Sikkema et al. 1995). As follows from the work (Wongsa et al. 2004), the rates of utilization of separate oil fractions may be significantly differed even in case of one and the same strain of hydrocarbon-oxidizing microorganisms. As a result, the influence of microorganisms on crude oil in soil may be accompanied by substantial changes in the initial composition of hydrocarbons, while the rest of hydrocarbons in soil may have absolutely different properties compared to the initial characteristics. The term 'waste oil'

The Waste Oil Resulting from Crude Oil Microbial Biodegradation in Soil 71

and Kuzyakov 2008). Obviously, unambiguous determination of PE by CO2 production calls for an exogenous substrate different from SOM in carbon isotopes (Zyakun et al. 2003; Dilly and Zyakun 2008; Zyakun et al. 2011). It has been shown that addition to the soil of a substrate easily accessible for microorganisms (e.g., glucose, amino acids, etc.) (Harabi and Bartha1993; Shen and Bartha 1996; Zyakun and Dilly 2005; Blagodatskaya and Kuzyakov 2008), contributes to the increase of SOM mineralization rate 2-3-fold compared to the processes in native soil. Acceleration of SOM degradation (positive PE) was also observed in case of addition of an aliphatic hydrocarbon (n-hexadecane) to the soil. Introduction into soil of n-hexadecanoic acid, the product of n-hexadecane oxidation, resulted in the lower rate of SOM mineralization compared to native soil (negative PE) (Zyakun et al. 2011). In the light of brief presentation of methods characterizing biodegradation and transformation of exogenous organic products entering the soil, the fate of crude oil in soils may be defined by the following parameters: (a) the rate of CO2 production as result of mineralization of crude oil and SOM; (b) activation of mineralization of native soil organic matter by introduced substrate (priming effect); c) the ratio of the quantities of biomass of the microorganisms growing on oil hydrocarbons as a substrate and quantities of SOM mineralized into CO2.

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

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>

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

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

CO2(t)=K+r·exp(µ·t) (1)

**2.1 CO2 sampling** 

production and carbon isotope analysis.

**2.2 The kinetics of CO2 respiration** 

parameters of equation [1]:

from ATP generation.

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 and Kuzyakov 2008). Obviously, unambiguous determination of PE by CO2 production calls for an exogenous substrate different from SOM in carbon isotopes (Zyakun et al. 2003; Dilly and Zyakun 2008; Zyakun et al. 2011). It has been shown that addition to the soil of a substrate easily accessible for microorganisms (e.g., glucose, amino acids, etc.) (Harabi and Bartha1993; Shen and Bartha 1996; Zyakun and Dilly 2005; Blagodatskaya and Kuzyakov 2008), contributes to the increase of SOM mineralization rate 2-3-fold compared to the processes in native soil. Acceleration of SOM degradation (positive PE) was also observed in case of addition of an aliphatic hydrocarbon (n-hexadecane) to the soil. Introduction into soil of n-hexadecanoic acid, the product of n-hexadecane oxidation, resulted in the lower rate of SOM mineralization compared to native soil (negative PE) (Zyakun et al. 2011). In the light of brief presentation of methods characterizing biodegradation and transformation of exogenous organic products entering the soil, the fate of crude oil in soils may be defined by the following parameters: (a) the rate of CO2 production as result of mineralization of crude oil and SOM; (b) activation of mineralization of native soil organic matter by introduced substrate (priming effect); c) the ratio of the quantities of biomass of the microorganisms growing on oil hydrocarbons as a substrate and quantities of SOM mineralized into CO2.
