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1 Introduction

Organochlorine pesticides have been used extensively all over the world for public health and agricultural purposes. Currently, their use is being phased out because of their toxicity, environmental persistence, and accumulation in the food chain. Hexachlorocyclohexane (HCH) is one of the most extensively used organochlorine pesticides for both agriculture and medical purposes. Though the use of technical mixture containing eight stereoisomers was banned in several advanced countries in the 1970s, many developing countries continue to use lindane (γ-HCH) for economic reasons. Thus, new sites are continuously being contaminated by γ-HCH and its stereoisomers (Blais et al. 1998; Kidd et al. 2008; Fuentes et al. 2010). Although only lindane is insecticidal, HCH as a group is toxic and considered as potential carcinogens (Walker et al. 1999).

For the supply of γ isomer, the other stereoisomers are separated from γ-HCH and dumped as waste at different spots on the production sites causing serious soil pollution (Li 1999). HCH continues to pose a serious toxicological problem at industrial sites where post-production of lindane along with unsound disposal practices has led to serious contamination, and HCH contamination continues to be a global issue (Kidd et al. 2008). These compounds have moderate volatility and can be transported by air to remote locations (Galiulin et al. 2002). Therefore, a possible pathway for bioremediation of contaminated soils is the use of indigenous microorganisms. It is known that the microbial degradation of chlorinated pesticides such as HCH is usually carried out by using either pure or mixed culture systems. There have been some reports regarding aerobic degradation of γ-HCH by Gram-negative bacteria like Sphingomonas (Singh et al. 2000; Kidd et al. 2008) and by the white-rot fungi Trametes hirsuta, Phanerochaete chrysosporium, Cyathus bulleri, and Phanerochaete sordida (Mougin et al. 1999; Singh and Kuhad 2000). However, little information is available on the ability of organochlorine pesticide biotransformation by Gram-positive microorganisms and particularly by actinomycete species, the main group of bacteria present in soils and sediments (De Schrijver and De Mot 1999). These Gram-positive microorganisms have a great potential for biodegradation of organic and inorganic toxic compounds and also could remove different organochlorine pesticides when other carbon sources are present in the medium as energy source (Ravel et al. 1998; Amoroso et al. 1998; Benimeli et al. 2003). Therefore, the ability of actinomycetes to transform organochlorine pesticides has not been widely investigated, despite studies demonstrating that actinomycetes, specifically of the genus Streptomyces, have been able to oxidize, partially dechlorinate, and dealkylate aldrin, DDT, and herbicides like metolachlor or atrazine (Ferguson and Korte 1977; Radosevich et al. 1995). In addition to their potential metabolic diversity, strains of Streptomyces may be well suited for soil inoculation as a consequence of their mycelial growth habit, relatively rapid rates of growth, colonization of semi-selective substrates, and their ability to be genetically manipulated (Shelton et al. 1996). One additional advantage is that the vegetative hyphal mass of these microorganisms can differentiate into spores that assist in spread and persistence.

Recent studies demonstrate significantly enhanced dissipation and/or mineralization of persistent organic pollutants (such as organochlorine pesticides; Kidd et al. 2008) at the root–soil interface or rhizosphere (rhizodegradation) (Kuiper et al. 2004; Chaudhry et al. 2005; Krutz et al. 2005). This rhizosphere effect is generally attributed to an increase in microbial density, diversity and/or metabolic activity due to the release of plant root exudates, mucigel, and root lysates (Curl and Truelove 1986). A summary of potential root zone carbon sources is given in Table 10.1. Rhizodeposits not only provide a nutrient-rich habitat for microorganisms but can potentially enhance biodegradation in different ways: they may facilitate the co-metabolic transformation of pollutants with similar structures, induce genes encoding enzymes involved in the degradation process, increase contaminant bioavailability, and/or selectively increase the number and activity of pollutant degraders in the rhizosphere (Schnoor et al. 1995; Burken and Schnoor 1998; Miya and Firestone 2001; Shaw and Burns 2003).

Table 10.1 Chemical compound observed in plant root exudates and extracts
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The objective of this chapter is to study the bioremediation capacity of indigenous actinomycete strain and the effect of root exudates of Zea mays on this process.

2 Lindane Removal by Streptomyces sp. M7 in a Soil Extract Medium

Benimeli et al. (2007a) studied the growth of Streptomyces sp. M7 in a soil extract medium (SE) with and without lindane addition. Carbon and nitrogen composition of SE were 0.5 and 0.01 g L−1, respectively. Nevertheless and despite the poor organic matter in SE, Streptomyces sp. M7 was able to grow in this medium for limited time.

When the effect of the temperature (25, 30, and 35 °C) on the growth of Streptomyces sp. M7 in SE was analyzed, it was observed that 25 °C was the optimal temperature of microbial growth. When Streptomyces sp. M7 was cultured in SE supplemented with lindane 100 μg L−1, at different incubation temperatures (Fig. 10.1), a maximum growth of 0.11 mg mL−1 was observed at 25 °C. Significant differences in the biomass were not observed at 30 and 35 °C. These results would indicate that the optimal temperature for the growth of Streptomyces sp. M7 in SE, in presence as well as in absence of the pesticide, is 25 °C.

Fig. 10.1
figure 00101

Effect of temperature on the bacterial growth of Streptomyces sp. M7 in SE medium amended with lindane 100 μg L−1. 25 °C (filled squares), 30 °C (filled circles), and 35 °C (filled inverted triangles). Bars represent standard deviations

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It is important to observe that the presence of lindane in culture medium did not inhibit the growth of Streptomyces sp. M7, since significant differences in bacterial growth in SE with and without the pesticide were not observed (p > 0.05). Similar results were obtained previously (Benimeli et al. 2007b), when Streptomyces sp. M7 was cultured in minimal medium supplemented with lindane 100 μg L−1, suggesting that the pesticide could not be toxic for this microorganism and that would not either be accumulated toxic intermediary metabolites that had an inhibiting effect on the growth.

Streptomyces sp. M7 was able to grow in SE over a relatively wide range of initial pH. No significant differences were observed when the microorganism was cultured at initial pH of 5, 7, or 9 (p > 0.05). When Streptomyces sp. M7 was cultured in SE added with lindane 100 μg L−1, at different initial pH, a maximum growth of 0.06 mg mL−1 was observed at pH 7; nevertheless, the microorganism was not able to grow at pH 5 and 9 (Fig. 10.2). The obtained results would indicate that the optimal initial pH for the growth of Streptomyces sp. M7 in SE with lindane is 7.

Fig. 10.2
figure 00102

Effect of pH on the bacterial growth of Streptomyces sp. M7 in SE amended with lindane 100 μg L−1. pH 5 (stars), pH 7 (filled triangles), and pH 9 (filled diamonds). Bars represent standard deviations

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Figure 10.3 shows the impact of the incubation temperature on the lindane removal by Streptomyces sp. M7. The maximum lindane removal was 70.4 % when the microorganism was incubated in SE at 30 °C. Although the optimum temperature for Streptomyces sp. M7 growth, with and without lindane, was 25 °C, the optimal temperature for the pesticide removal was 30 °C.

Fig. 10.3
figure 00103

Effect of temperature on the lindane removal by Streptomyces sp. M7 in SE amended with lindane 100 μg L−1. 25 °C (filled squares), 30 °C (filled circles), and 35 °C (filled inverted triangles). Bars represent standard deviations

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Bachmann et al. (1998) reported that temperature of 30 °C was most favorable for the biodegradation of lindane in soil slurry by the mixed native microbial population of the soil. Arisoy and Kolankaya (1997) observed that the suitable incubation temperature for maximum growth and degradation activity of lindane by the fungus Pleurotus sajor-caju was 30 °C. Manonmani et al. (2000) also observed the degradation of the γ-HCH isomer by a microbial consortium under a wide range of temperatures (4–40 °C) in a liquid culture medium, and 30 °C was the optimum for γ-HCH degradation. Siddique et al. (2002) obtained similar results studying the effect of incubation temperature in the biodegradation of lindane by Pandoraea sp.; an incubation temperature of 30 °C was optimum for degradation of γ-HCH (57.7 %) in liquid culture and soil slurry (51.9 %).

The effect on lindane removal by Streptomyces sp. M7 in SE at initial pH of 5, 7, and 9 is presented in Fig. 10.4. Removal of pesticide (47.2 and 38.0 %) was observed at initial pH of 5 and 9, respectively, at 28 days of incubation. The highest removal ability of Streptomyces sp. M7 (70.4 %) was noted at an initial pH = 7 at 28 days of incubation. The fate of organic pollutants in the environment is influenced by environmental factors, such as pH and temperature, affecting the activity of microorganisms. Streptomyces sp. M7 was able to remove lindane over a wide range of pH in SE.

Fig. 10.4
figure 00104

Effect of pH on the lindane removal by Streptomyces sp. M7 in SE amended with lindane 100 μg L−1. pH 5 (stars), pH 7 (filled triangles), and pH 9 (filled diamonds). Bars represent standard deviations

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Arisoy and Kolankaya (1997) reported that medium pH = 5 was the optimum for both growth and degradation activity of lindane by the fungus Pleurotus sajor-caju. Manonmani et al. (2000) examined the influence of pH on the degradation of the γ-HCH isomer in a basal mineral medium by an acclimated consortium of microorganisms. They found that a pH range of 6–8 was most favorable for growth and degradation of the pesticide. Siddique et al. (2002) reported that Pandoraea species showed the highest degradation of α- and γ-HCH at an initial pH of 8 in broth culture.

3 Lindane Removal by Streptomyces sp. M7 in Sterile Soil Samples

Benimeli et al. (2008) studied the growth of Streptomyces sp. M7 in sterile soil samples by adding different lindane concentrations in 4 weeks. Simultaneously, lindane removal by Streptomyces sp. M7 was determined by gas chromatography; similar experiments were carried out without lindane as controls. As shown in Fig. 10.5, the cell concentration increased during incubation and significant differences in the growth were not observed at different lindane concentrations added, as in the control without lindane. These results would indicate that the growth of Streptomyces sp. M7 in soil was not affected by the lindane concentrations assayed, suggesting that the microorganism could tolerate or may degrade the pesticide by producing the necessary dehalogenase enzymes as was demonstrated by Nagata et al. (1999) for Sphingomonas paucimobilis UT26.

Fig. 10.5
figure 00105

Streptomyces sp. M7 growth in sterile soil samples, during 4 weeks of incubation, without (open squares) and with 100 (open squares), 150 (filled circles), 200 (filled triangles), and 300 (filled diamonds) μg g−1 of lindane. Bars represent standard deviations

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Influence of different initial concentrations of lindane on pesticide removal was evaluated by determining residual lindane in the soil samples. At initial lindane concentrations of 100, 150, 200, and 300 μg kg−1, removal of pesticide after 4-week incubation was 29.1, 78.0, 38.8, and 14.4 %, respectively. Lindane concentrations in uninoculated control soils were unchanged after 4 weeks of incubation. This inhibition in the removal ability of Streptomyces sp. M7 could be due to the toxicity of metabolites, which might have former but not detected in this study by the methods employed. For demonstrating that, it could be necessary to measure the intermediary metabolites of lindane degradation as was determined by Nagata et al. (1999) for Sphingomonas paucimobilis UT26.

Awasthi et al. (2000) found that at low initial concentrations of endosulfan in soil (50 and 100 μg g−1 soil), the degradation was very rapid in inoculated soils; at higher concentrations, the degradation rates were slower, leading to a total inhibition of the degradation activity at the initial concentration of 10 mg g−1. Similar findings were reported by Okeke et al. (2002), who studied the ability of a Pandoraea sp. strain to remove lindane in liquid and soil slurry cultures. Their results indicated that the rates and extent of lindane removal increased with increasing concentrations up to 150 mg L−1 but declined at 200 mg L−1, after 4–6 weeks of incubation. However, in a similar study, Liu et al. (1991) found opposite results when they inoculated a Streptomyces sp. strain in sterile soil samples spiked with metolachlor and observed that the amount of residual metolachlor was higher at lower herbicide concentrations, after 1-week incubation.

This result indicates that Streptomyces sp. M7 could remove lindane from soil but at limited pesticide concentration and the viable bacterial count of the soil culture indicated growth and survival of Streptomyces sp. M7. This is not surprising because the soil contains available organic nutrients that the bacteria may prefer for growing. Then, lindane could be used as a secondary substrate source as it was demonstrated previously by Benimeli et al. (2007b) in culture medium where glucose at limited concentration was added.

To arrive at the optimal number of bacterial cells for effective removal of lindane, the influence of different inoculum sizes (0.5–4.0 g kg−1 ww soil) was studied in sterile soil samples with lindane 100 μg kg−1 ww soil (Fig. 10.6). 28 days after inoculation with different Streptomyces sp. M7 concentrations into lindane-amended autoclaved soil, the cell population increased rapidly in 2 weeks and was followed by a stationary phase from 2 to 4 weeks. This growth profile was observed for all assayed bacterial concentrations and was similar in contaminated as well as non-contaminated soil samples. These results reinforce the hypothesis according to which lindane concentrations present in soil were not toxic for Streptomyces sp. M7. The inoculum 2 g cells kg−1 soil (ww) significantly led the maximum bacterial count (9.0 × 1012 CFU kg−1 ww of soil).

Fig. 10.6
figure 00106

Effect of inoculum size on the growth of Streptomyces sp. M7 in sterile soil samples, during 4 weeks of incubation. Symbols used: (triangles) 0.5 g kg−1 soil, (stars) 1.0 g kg−1 soil, (open squares) 2.0 g kg−1 soil, and (open circles) 4.0 g kg−1 soil

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A substantial decline of the residual lindane at different inoculum concentrations was observed within 0–2 weeks of incubation, whereas the compound did not disappear from the uninoculated sterile control. However, the percentage of lindane removal was not entirely proportional to the amount of Streptomyces sp. M7 initially added to the soils, indicating that the two parameters are not in direct proportion. Maximal pesticide depletion (56.0 %) was observed at 2 g cells kg−1 soil (ww) and thereafter decreased at 4 g cells kg−1 soil (Table 10.2).

Table 10.2 Effect of inoculum size on the lindane removal by Streptomyces sp. M7 in sterile soil samples, after 4 weeks of incubation. Lindane initial concentration: 100 μg kg−1 ww soil
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In a similar work, Liu et al. (1991) found that approximately 80 % of the pesticide metolachlor was transformed in a sterile soil inoculated with a Streptomyces sp. strain after 1 week of incubation; however, the rate of metolachlor transformation was not proportional to the inoculum size. Kumar-Ajit et al. (1998) studied the inoculation of 3-chloro and 4-chlorobenzoate-treated sterile soil with a chlorobenzoate-degrading Pseudomonas aeruginosa 3mT; they probed three inoculum sizes (1, 2, and 4 mg cells kg−1 soil), and these were effective in degrading the chemical in the soil, but the degradation was faster with a larger inoculum. On the other hand, Johri et al. (2000) found that an increase in the inoculum size of Sphingomonas paucimobilis resulted in an increase in the degradation rate of the HCH isomers in the culture medium, indicating that the two parameters are in direct proportion.

4 Bioremediation of Lindane-Contaminated Soil Samples by Streptomyces sp. M7 and Effect of Pesticide on Maize Plants

Nonsterile soil spiked with lindane 100 μg kg−1 was inoculated with 2 g kg−1 Streptomyces sp. M7, and after 14 days incubation, Zea mays seeds were grown in this soil. Concentrations of residual lindane in soil were monitored periodically to assess the effectiveness of the inoculated strain in the bioremediation of the soil.

A strong decrease in the residual concentration of added lindane was observed in Streptomyces sp. M7-inoculated soil. After 14 days of incubation, 68 % of lindane was removed (Fig. 10.7). There were no evident changes in the concentration of the pesticide in the uninoculated soil, indicating that the native microorganisms were not involved into the pesticide removal.

Fig. 10.7
figure 00107

Removal of 100 μg kg−1 of lindane by Streptomyces sp. M7 in non-sterile soil samples, during 4 weeks of incubation. 2.0 g cells (ww) kg−1 soil was added as inoculum. Symbols used: (open squares) residual lindane in uninoculated control soil, (filled squares) residual lindane in inoculated soil

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In soil bioremediated with Streptomyces sp. M7, normal germination (100 %) and an increased in the seedling vigor were observed, compared to the control maize seeds (Table 10.3). It is possible that the pesticide was removed by the inoculated microorganism before seed germination began and the involvement of indigenous microflora capable of metabolizing lindane was not observed.

Table 10.3 Growth, seed germination, and seedling vigor of maize seeds in lindane bioremediated soil by Streptomyces sp. M7
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In a similar study by Krueger et al. (1991), soybean and pea seedlings susceptible to the herbicide dicamba were protected from its deleterious effect by inoculating soils with dicamba-degrading microorganisms. Kumar-Ajit et al. (1998) demonstrated that chlorobenzoates adversely affect the seed germination and seedling vigor of tomato. However, the bioremediation of the soil with Pseudomonas aeruginosa 3mT protected the tomato seeds, resulting in the normal germination and seedling vigor. Bidlan et al. (2004) observed that the effect of tech-HCH on germinating radish and green gram seeds was nullified by treatment of contaminated soil with a HCH-degrading microbial consortium.

Although no maize plant growth promotion was observed when Streptomyces sp. M7 was inoculated into the contaminated lindane soil samples, the fact that this pesticide can be removed and therefore its uptake by the plant into biomass could be lowered, avoiding the contamination through the food chain. The low water solubility and high hydrophobicity of lindane make their uptake and translocation within the plant unlikely (Kidd et al. 2008).

5 Lindane Removal by Streptomyces sp. M7 Cultured on Medium Amended with Root Exudates of Maize

Root exudates are products released in the root zone of plants with significant levels of carbon (and possibly nitrogen). The exudates are likely to favor fast-growing microbes, provided they have the corresponding metabolic abilities. In this context, Alvarez et al. (2010; Alvarez et al. 2012) studied the growth of Streptomyces sp. M7 in minimal medium supplemented with root exudates of maize and/or glucose (0.8 g L−1) and spiked with lindane (1.66 mg L−1). Cultures were incubated at 30 °C at 150 rpm for 7 days. Similar experiments were carried out without lindane as controls. Root exudates were collected from solution cultures of maize according to Luo et al. (2006). Exudates were lyophilised and stored at 4 °C until sample processing.

Regarding composition, carbohydrate and organic carbon of root exudates were 41.6 mg g−1 and 40 mg g−1, respectively. The highest level of pesticide removal was obtained when Streptomyces sp. M7 was cultured in presence of root exudates (Table 10.4). In contrast, although biomass production was highest in the glucose treatment, the percentage of lindane removal under this condition was lower.

Table 10.4 Biomass production and lindane removal by Streptomyces sp. M7 after 7 days of growth on minimal medium supplemented with different carbon sources
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An increase in degradation of a wide range of organic pollutants was observed as result of modified microbial activity in the rhizosphere of several plants (Pandya et al. 1999; White 2001; Chekol et al. 2002; Corgié et al. 2003; Muratova et al. 2003; Shaw and Burns 2005; Phillips et al. 2005). Enhanced transformation of the explosive trinitrotoluene (TNT) by the forage grasses Phalaris arundinacea and Panicum virgatum was shown by Chekol et al. (2002). Concentrations of the organochlorine p,p′ DDE (2,2-bis (p-chlorophenyl) 1,1-dichloroethylene), a metabolite of DDT, were significantly reduced in the rhizosphere of field-grown zucchini, pumpkin, and spinach compared to bulk soil (White 2001). In contrast, phenanthrene-degrading activity of Pseudomonas putida ATCC 17484 was repressed after incubation with plant root extracts (Rentz et al. 2004, 2005). However, these authors suggested that the enhanced microbial growth on rhizodeposits is likely to compensate for this partial repression since a larger microbial population leads to a faster degradation rate.

Little is known about the mechanisms involved in interaction of root exudates with bacteria. In this respect, Chen and Aitken (1999) and Hegde and Fletcher (1996) found that terpenes (such as cymene, α-pyrene, and α-terpinene) and phenolics (such as salicylate) released by roots of certain plants induced biphenyl dioxygenase in polychlorinated biphenyls-degrading bacteria. It could be possible that root exudates of maize enhanced removal of lindane by Streptomyces sp. M7 by means of a similar mechanism.

6 Lindane and Metabolite Determination in Cell-Free Extract by GC-Mass Spectrometry Analysis

The first signs of the aerobic lindane degradation were determined by Nagata et al. (1999). It was demonstrated that Sphingobium japonicum UT26 possesses a dechlorinase enzyme, LinA (γ-hexachlorocyclohexane dehydrochlorinase, EC 4.5.1), encoded by the linA gene that catalyzes two dehydrochlorination steps: γ-HCH to 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN) via γ-pentachlorocyclohexene (γ-PCCH). In addition to γ-HCH and γ-PCCH, α- and δ-isomers of HCH were also dehydrochlorinated by LinA, whereas β-HCH was not (Nagata et al. 1993). Furthermore, it was experimentally confirmed that dehydrochlorination of γ-HCH proceeds by a 1,2-ante dehydrochlorination reaction (Nagata et al. 2007).

Regarding the environmental problems caused by lindane and the current lack of information about the presence of dechlorinase activity in Streptomyces, the aim of this point was to demonstrate, for the first time, a specific dechlorinase activity in Streptomyces using lindane as substrate. In order to determine lindane and metabolites in cell-free extract of Streptomyces sp. M7, the strain was grown in flasks with 250 mL of MM containing γ-HCH 100 μg mL−1 and incubated at 30 °C at 100 rpm for 48 and 96 h. At the beginning of the experiment, the inoculum was 150 μl of concentrated spore suspension (109 CFU mL−1). Supernatants were extracted by solid phase extraction (SPE) using C18 columns, evaporated to dryness under reduced pressure, and the residue was resuspended in hexane. Routine quantitative determinations of lindane (γ-HCH) γ-pentachlorocyclohexene (γ-PCCH) and 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN) were carried out with gas chromatograph-micro-electron capture detection (GC-μECD) (Cuozzo et al. 2009).

The gas chromatography results of the cell-free extracts obtained at 48 and 96 h of growth of Streptomyces sp. M7 revealed the appearance of γ-PCCH (Rt 6.26 min) and 1,4-TCDN,(Rt 5.29 min), the first and second product of the lindane catabolism by the specific dechlorinase in the catabolic way proposed by Nagata et al. (2007). The relative abundance of γ-PCCH and the 1,4-TCDN increased one and half times, at 96 h compared to 48 h of growth (Table 10.5).

Table 10.5 Relative abundance analysis by GC-Mass of γ-HCH and intermediates γ-PCCH and 1,4-TCDN during aerobic degradation of γ-HCH by Streptomyces sp. M7
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However, these results indirectly demonstrated the presence of one specific enzyme in the lindane degradation way from Streptomyces sp. M7. It is the first report on dehalogenase activity in actinomycetes with lindane as specific substrate. This has only been reported in Sphingomonas (Nagata et al. 2007; Normand et al. 2007) detected a putative 2,5-dichloro-2,5cyclohexadiene-1,4-diol dehydrogenase (2,5-DDOL dehydrogenase) in Frankia. Genetic studies of this strain are necessary for a proper understanding of the principle of its ability to degrade different chlorinated hydrocarbon compounds.

7 Conclusions and Perspectives

The review clearly suggests that Streptomyces sp. M7 has the capacity to grow in a soil extract broth, a nutritionally poor medium, in the presence of lindane, and to remove the pesticide. Since streptomycetes are metabolically diverse and relatively resistant to adverse conditions that may occur in the soil environment, Streptomyces sp. M7 could be considered as attractive targets for lindane degradation in situ. In addition, the development of bioremediation processes using indigenous microorganisms is advantageous, as strains isolated are already adapted to the substrate, and to local soil and climatic conditions, and regulatory and legislation issues are simpler, compared to the introduction and release of exogenous microorganisms and genetically modified organisms into the environment. On the other hand, Streptomyces sp. M7 bioremediation activity is not inhibited by the natural soil microbial flora. Moreover, Streptomyces sp. M7 growth was not inhibited by 300 μg kg−1 of lindane.

Regarding the use of plants (or their products) for the treatment of contaminated sites, preliminary results suggest that Streptomyces sp. M7 was stimulated by the root exudates of maize amended to the culture. In this context, phytostimulation may be a promising strategy for the remediation of HCH-contaminated soils. It is important to continue these studies not only at the laboratory but also in field trails in order to evaluate the potential of Streptomyces sp. M7 in the bioremediation of natural habitats. Further studies evaluating the soil–plant–microbe system and its influence on HCH biodegradation are necessary so as to better explore and exploit an undoubtedly huge potential.