Análisis de la Competencia

Development of the Chemical Industry over the past few decades has resulted in production and application of new chemical compounds which now can be found in a range of everyday use products ranging from pharmaceuticals (Vieno et al., 2006) to personal care (Ramirez et al., 2009) and household products (Fenet et al., 2003). Due to the nature of their application, such chemicals enter the environment primarily via wastewater. In order to determine the persistence and potential toxic effects of chemicals in the environment, standardized biodegradation tests have been established by the Organisation for Economic Cooperation and Development (OECD).

These tests are usually conducted under environmentally unrealistic conditions. For instance, light is excluded from tests or diffuse light is recommended in OECD biodegradation tests to avoid any potential test failure caused by overgrowth of algae. Light is a major concern in biodegradation studies and particularly in tests that are using respirometric methods to assess the biodegradation of chemicals, where interference from increased dissolved oxygen may occur due to the photosynthesis conducted by algae. Dissolved oxygen also affects aerobic chemical biodegradation, since oxygen serves as an electron acceptor in the biodegradation processes (Xu et al., 2005). Algae developing under light conditions may also compete with bacteria for the energy source, and may impact biodegradation kinetics of test chemicals. Light is also responsible for direct and indirect photolysis of chemicals. Direct photolysis is light-dependent degradation of chemicals while indirect photolysis is caused by light-induced growth of phototrophs which are capable of chemical biodegradation. Light can induce radicals which are involved in oxidation of nitrophenols (Kiwi et al., 1994). Additionally, light in combination with sensitizers

58 can stimulate the photodegradation of chlorophenols (Bandara et al., 2001). Light is responsible for photochemical reactions which occur near the water surface e.g. in river water (Klöpffer, 1992) where chemicals may undergo photo-oxidative degradation (Datta et al., 2004) and photocatalytic degradation (Li et al., 2005). Interestingly, light conditions may also favour the biodegradation of chemicals by phototrophs i.e. algae may influence photolysis rates of chemicals in water. This is due to the light induced algal photosynthesis which results in enhanced algal growth. Zepp and Schlotzhauer (1983) reported that from the group of 22 compounds that they studied accelerated light-dependent degradation of certain organic chemicals was observed in the presence of green and cyanobacteria at concentration of 1-10 mg of chlorophyll a/L.

Phototrophic microorganisms have been reported as capable of chemical biodegradation under light conditions by Roldán et al. (1998) who demonstrated the degradation of PNP by the bacterium Rhodobacter capsulatus which was incubated in the presence of light. Two species of microalgae, Chlorella vulgaris var. vulgaris

f. minuscula and Coenochloris pyrenoidosa were isolated by Lima et al. (2003) from microalgal consortium which degraded PNP. Both were capable of PNP degradation when cultured separately. Hirooka et al. (2006) reported that from the mixture of photoautotrophic microorganisms two microorganisms Chlamydomonas reinhardtii

and Anabaena cylindrical had the highest ability to remove 2,4-dinitrophenol (2,4- DNP) and 2-amino-4-nitrophenol (2-ANP) in liquid medium under light conditions. Algae are also capable of biodegradation of aromatic chemicals in the mixed algal- bacterial culture systems (Borde et al., 2003). These results prove that algae can also play a key role in the biodegradation of chemicals present in surface water, and therefore, should not be excluded in the biodegradation tests.

Absence of light is not the only factor which makes OECD biodegradation tests unrealistic. The amount of chemical applied in biodegradation tests may cause difficulties with data interpretation and extrapolation of results to environmental compartments. In wastewater treatment or in the environment many chemicals are present at ng/L to μg/L levels whereas in the OECD tests the concentrations are at the level of mg/L (Ahtiainen et al., 2003). The reason biodegradation tests have developed in this manner is that dosing at realistic concentrations requires labelled compounds and specific analysis, and therefore, more convenient approach was

59 developed for environmental risk assessment of thousands of compounds. Kinetics of biodegradation of chemicals and chemical bioavailability depend on chemical concentration. Biodegradation of a chemical at higher concentrations is usually a growth-linked process (Rhee et al., 2002; Abuhamed et al., 2004) whereas at low concentrations chemicals do not serve as primary substrates in the environment where a variety of compounds are present (Ramakrishnan et al., 2011). Therefore, cometabolism may exist at lower concentrations of the primary compound which results in lower chemical biodegradation rates (Wang et al., 1984; Novic and Alexander, 1985). In addition, very low chemical concentrations affect the rate of entry of substrate into the cell (Battersby, 1990), and since many microbial transporters and catabolic enzymes are regulated and are synthesized in response to the presence of a certain concentration of their substrate (Harms and Bosma, 1997) the induction of enzymes which are involved in the biodegradation pathways of test compounds might require higher concentration of chemical (Hanne et al., 1993). Application of high levels of test compound can be useful in identifying populations that biodegrade the compound and can also be used to assess differences in biodegradation potential of inocula from different environmental compartments (Johnson et al., 2004). However, high chemical concentration cannot be used for the prediction of chemical biodegradation under environmental conditions, since, for reasons explained above, biodegradation may not reflect the process under typical environmental conditions. Moreover, false negative test results may be generated with high concentrations if these are toxic to the microbial inoculum (Comber and Holt, 2010). Hence, realistic concentrations of chemical should be introduced into biodegradation systems for better understanding of processes occurring under environmentally relevant conditions.

The most direct approach for identifying chemical-degrading consortia is by analysis of functional genes that encode key enzymes in degradation of such chemicals. Some authors have already applied the functional gene approach to their biodegradation studies. Piskonen et al. (2008) used naphthalene dioxygenase and toluene dioxygenase genes for monitoring efficiency of toluene and naphthalene biodegradation in soil and Zhou et al. (2006) investigated diversity of bacteria degrading polycyclic aromatic hydrocarbons in mangrove sediments, using dioxygenases as functional markers. In the current project, para-nitrophenol (PNP)

60 was chosen as a model compound and procedures were developed to use genes involved in PNP biodegradation pathways as functional markers to study PNP- degrading bacterial populations in biodegradation experiments inoculated with environmental communities.

In addition, application of emerging high throughput sequencing methods in combination with the functional gene analysis was used in this project. High throughput sequencing is a powerful technique which enables fast generation of a large amount of sequencing data from samples. These methods have been reported as useful tools to study in depth populations involved in biodegradation processes (Iwai

et al., 2010; Warnecke and Hess, 2009), tracking shifts in distribution and structure of microbial communities caused by a variety of environmental factors (Acosta- Martínez et al., 2008, Mackelprang et al., 2011), linking the community structure and function (Fuhrman, 2009) and detection and identification of uncultivable degraders (Eyers et al., 2004; Chen and Murrell, 2010).

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