Análisis del mercado y sus segmentos

The choice of a suitable model compound for biodegradation tests depends on its solubility in water, resistance to microbial biodegradation, and potential environmental impacts. Although para-nitrophenol (PNP) does not belong to DTD chemicals, it is used here as a model compound to conduct experiments which address general questions regarding chemical biodegradation in the environment to later inform specific studies. PNP is a good model compound since it is present in the environment, it has been used in biodegradation tests and there is available information on pathways and functional genes involved in PNP biodegradation. 1.9.1. PNP in the environment

Nitroaromatic compounds, including nitrophenols, are widely distributed in environment, and among these chemicals, para-nitrophenol (PNP) (Figure 1.5 a and 1.5 b) is one of the most important both in terms of quantities used and potential

45 environmental impacts (ATSDR, 1992; Qiu et al., 2007). It is a manufactured chemical which does not occur naturally in the environment and the primary anthropogenic source of PNP in the air is probably industrial manufacturers. Very little of this compound is directly released to surface water or soil. Like other nitroaromatic compounds, it can be formed as a result of atmospheric photochemical reactions of several aromatic compounds from anthropogenic sources (ATSDR, 1992; Calza et al., 2008). It can be also found in vehicular exhausts as a result of the thermal reaction of fuel with oxides and nitrogen. Additionally, PNP is formed as a degradation product and it is an impurity in parathion formulation (ATSDR, 1992).

a b

Figure 1.5: Para-nitrophenol.

Where: a-Chemical structure of para-nitrophenol, source: ChemSpider, 2012; b-physical appearance

of para-nitrophenol, source: Wikipedia, 2012 a.

1.9.2. Application and properties of para-nitrophenol

PNP has application in agriculture, dyes, pigments, engineering polymers and pharmaceuticals. It is also used as fungicide for leather, production of parathion and other organic synthesis (Montgomery and Welkon, 1955; Cho et al., 2000; Qiu et al.,

2007). PNP is a major urinary metabolite of parathion and can be used as a biomarker of human exposure to pesticides since it is a breakdown product of parathion and fluoridifen (Rogers and Emon, 1993). PNP has a good solubility in water (Table 1.6), the log Kow of 1.9 and vapor pressure of 9.86 x 10-2 Pa (Comber and Holt, 2010). It is toxic to plant, animal and human health, with mutagenic and carcinogenic activities. The purification of wastewaters contaminated with nitroaromatic chemicals is very difficult since they are resistant to conventional

46 treatment techniques (Kuşçu and Sponza, 2009). In air, destruction of PNP is mainly due to photolysis and physical removal processes such as gravitational settling of aerosols and wet deposition by rain and snow. In water, both photolysis and biodegradation take part in removal of PNP; photolysis is important in near surface water. In soil the biodegradation of PNP is the most important fate process (ATSDR, 1992).

Table 1.6: Physico-chemical properties of para-nitrophenol.

Source: Wikipedia, 2012 a.

Properties

Molecular formula C6H5NO3

Molar mass 139.11 g/mol

Appearance Colorless or yellow pillars

Melting point 113–114 ˚C

Boiling point 279 °C, 552 K, 534 ˚F

Dissociation constant pKa 7.16 (22 ˚C)

Solubility in water

10 g/L (15 ˚C) 11.6 g/L (20 ˚C)

16 g/L (25 ˚C)

1.9.3. Para-nitrophenol as a model compound

PNP has been used as a model compound for chemical biodegradation studies by several authors (Ingerslev et al., 2000; Nyholm et al., 1996, Nyholm et al., 1992; Thouand et al., 1995) with variable mineralization results. The biodegradation of PNP can be rapid, but sometimes it occurs after a long lag phase. Toräng and Nyholm (2005) considered para-nitrophenol to be readily biodegradable after a lag phase. At 100 μg/L in natural surface waters they reported that the lag phase was reduced from 10 days to <1 day following an adaptation period of between one and five weeks. PNP is also used by Davenport et al., (2009) as a reference standard in their studies to investigate the importance of microbial density and diversity of inocula used in ready tests. Initial results indicated a high probability of

47 biodegradation (>70% parent compound degradation) with enhanced inocula concentrations and extended test duration (60 days) with low variability between inocula from six different locations for activated sludge and river water. Greater variation in the biodegradation of PNP was observed when a 28-day test was used. However, PNP is recommended as a reference chemical for the modified SCAS test (OECD 302A) (OECD, 1981b). It is also biodegradable in enhanced tests with increased biomass. Measured data (n=18) in non-standard tests suggest a median half-life for PNP of 2.5 days in the freshwater environment with a range from 1.3-77 days (Comber and Holt, 2010).

1.9.4. Biodegradation pathways and functional genes

There have been many reports on the biodegradation of PNP (Munnecke and Hsieh, 1974; Spain and Gibson, 1991; Roldán et al., 1998; Lima et al., 2003; Kulkarni and Chaudhari, 2006; Qiu et al., 2007). PNP biodegradation pathways, including genes encoding key enzymes involved in its biodegradation, are also well characterized (Kitagawa et al., 2004; Perry and Zylstra, 2007; Takeo et al., 2008; Zhang et al,

2009). Many PNP degrading bacterial strains belonging to the genera

Flavobacterium, Pseudomonas, Moraxella, Nocardia, and Arthrobacter have been isolated and their metabolic activity on PNP was defined. Two major degradation pathways of PNP (Figure 1.6) have been characterized (Chauhan et al., 2010; Kitagawa et al., 2004). Both pathways lead to degradation of PNP to maleylacetate but differ in the initial steps. PNP biodegradation via hydroquinone (hydroquinone pathway) has been found in Gram-negative bacteria such as Burkholderia spp., and

Moraxella spp. (Prakash et al., 1996; Spain and Gibson, 1991), whereas PNP biodegradation via 4-nitrocatechol (4-nitrocatechol pathway) has been found in Gram-positive bacteria such as Bacillus spp., and Arthrobacter spp. (Jain et al.,

48

Figure 1.6: Pathways for PNP degradation in bacteria.

Source: Chauhan et al., 2010.

PNP degradative genes pnpA and npdA2 are responsible for the initial reaction in PNP biodegradation pathways in Gram-negative and Gram-positive bacteria, respectively. Gene pnpA encodes para-nitrophenol 4-monooxygenase (PnpA), which degrades PNP into hydroquinine through para-benzoquinine, and npdA2 encodes

para-nitrophenol 2-monooxygenase (NpdA2), which degrades PNP into 4- nitrocatechol.

Maleylacetate reductase (MAR) is a part of both PNP degradation pathways (Figure 1.6) and is responsible for the NADH- or NADPH-dependent reduction of maleylacetate to 3-oxoadipate or substituted maleylacetates to substituted 3- oxoadipates (Seibert et al., 2004). Maleylacetate is also an intermediate in the biodegradation of a number of other chemicals (Figure 1.7) and generally leads to production of 3-oxoadipate which is converted to succinyl-CoA and acetyl-CoA (Harwood and Parales, 1996), which enter the tricarboxylic acid cycle (TCA). Maleylacetate reductase genes have been detected and described in many bacteria e.g. in Rhodococcus opacus 1CP (gene macA; Seibert et al., 1998), Alcaligenes

49

eutrophus JMP134 (gene tfdF; Kasberg et al., 1995) and the clcE gene from

Pseudomonas sp. strain B13 (Kasberg et al., 1997) and it could be involved in he biodegradation pathways of a variety organic chemicals (Seibert et al., 2004).

Figure 1.7: Involvement of maleylacetate reductase in bacterial catabolic pathways of aromatic compound degradation. Source: Seibert et al., 1993.