Introduction

Diesel contains many highly concentrated toxic materials, and diesel contamination can negatively influence soil microbes and plants, as well as contaminate groundwater, which may be used for drinking or agriculture. Microbes play an important role in mitigating these environmental problems as they can limit the mobility of petroleum contaminants (Gestel et al. 2003; Hong et al. 2005; Franco-Ramírez et al. 2007). The use of plant-based systems to remediate contaminated soils has become an area of intense scientific study in recent years and it is apparent that plants which grow well in contaminated soils need to be identified and screened for use in phytoremediation technologies. Adam and Duncan (2002) investigated the effect of diesel fuel on germination of selected plant species. Germination response varied greatly with plant species and was species specific, as members of the same plant family showed differential sensitivity to diesel fuel contamination (Adam and Duncan 2002). Since hydrocarbons are natural products, it is not surprising to find organisms that are able to degrade these energy-rich substrates (Singh and Lin 2008). Several studies have examined the relationship between plants and rhizosphere-based microbial communities. Phytoremediation systems were observed to increase the catabolic potential of the rhizosphere soil by altering the functional composition of the microbial community (Siciliano et al. 2003; Jaisoo et al. 2006). However, the specifics of how the rhizosphere enhances biodegradation are still unclear.

Trichoderma spp. are a group of filamentous fungi used extensively in agriculture as biofungicides and in industry as sources of several enzymes (Harman et al. 2004; Chulalaksananukul 2008). The genus Trichoderma is also able to colonize very different niches thanks to its metabolic versatility and to its tolerance to stress conditions. These properties make Trichoderma a widespread biocontrol agent of plant diseases. Some of the species are reported to be plant growth promoters and inducers of systemic resistance in plants (Harman et al. 2004). Fungal genera like Trichoderma are thought to be cellulase producers and crude enzymes produced by these microorganisms are commercially available for agricultural use. Recently an efficient producer of cellulases Trichoderma ressei has been reported (Chulalaksananukul 2008). In this report we present an investigation of the ability of T. ressei to promote growth of Chickpea (Cicer arietinum) in the diesel fuel contaminated soil.

Understanding how community processes affect ecosystem processes is a central challenge in ecology and microbial communities offer a potentially powerful opportunity for advancing this understanding, consequently, changes in the structure or function of microbial communities may have a major impact on ecosystem activities. Garland and Mills (1991) have described a method that allows observation of the functional potential of microbial communities using the Biolog system. This method shows the potential degrading abilities of a community in culture conditions. Other authors have demonstrated that these metabolic profiles can reveal differences between microbial communities that come both from different kinds of soils, and from different soil fractions (Winding 1994). Microbial communities have great potential for temporal or spatial change, and thus represent a powerful tool for understanding community dynamics in both basic and applied ecological contexts. A rapid, community-level approach for assessing patterns of sole carbon source utilization by mixed microbial samples has been used increasingly to study microbial community dynamics (Garland 1996; Gomez et al. 2000; Tripathi et al. 2006; Nautiyal et al. 2008; Sala et al. 2008). Present study describes changes in microbial activity and community composition during bioremediation of a soil sample contaminated with various amounts of diesel oil amended with T. ressei as bioinoculant in the chickpea rhizosphere.

Materials and methods

Plant growth

Effect of diesel on the germination of chickpea was determined for its ability to germinate in diesel fuel contaminated soil by adding 21.25 g of diesel/kg soil, representing low concentration of diesel (LCD) and 85 g of diesel/kg soil representing high concentration of diesel (HCD), as described earlier by Adam and Duncan (2002). Percentage germination observation was taken after 10 days. Plants were grown and maintained for 30 days as described earlier, before being harvested for analysis (Nautiyal et al. 2008). Chlorophyll content was measured as described earlier by Wellburn (1994).

Microbial analysis

Microflora associated with soil samples was determined by the culture enrichment technique (Nautiyal et al. 2008). The total microbial counts were determined as follows: Nutrient agar for heterogenous bacteria, Kenknight, and Munaier’s medium for Actinomycetes spp. and Rose Bengal Chloramphenicol agar for selective isolation of fungi (from HI-MEDIA Laboratories Pvt. Ltd., Bombay, India).

Soil analysis

Organic carbon was determined by dichromate oxidation (Nelson and Sommers 1982). Dehydrogenase activity was measured spectrophotometrically using characteristics of TTC (2,3,5-triphenyltetrazolium chloride) reduction to TPF (triphenylformazan) as described by Namkoong et al. (2002). Dehydrogenase activity was expressed in micrograms of formazan per gram of sample (mg-TPF/g sample).

Microbial diversity using carbon source utilization pattern

Biolog Eco plates (Biolog, Inc., Hayward, CA, USA) were used to determine the carbon source utilization pattern of chickpea rhizosphere samples. Root samples (1 g) was shaken in 99 ml of sterile 0.85% saline MQW for 60 min and then makeup a final dilution 10−3. After incubation 150 μl of sample were inoculated in each well of Biolog Eco plates and incubated at 30°C. The rate of utilization is indicated by the reduction of tetrazolium, a redox indicator dye, which changes from colorless to purple. Data were recorded for day 1–7 at 590 nm. Microbial activity in each microplate, expressed as average well color development (AWCD) was determined as described by Garland (1996). Diversity and evenness indexes were calculated as described by Nautiyal (2009). Principal component analysis (PCA) was performed on data divided by the average well color development (AWCD) as described by Garland and Mills (1991). Formulas used for diversity calculations were described by Nautiyal (2009), data collected after day 4 were used. Principal component analysis was performed on data divided by the average weighted color development (AWCD), as described by Garland and Mills (1991). Statistical analyses were performed using SPSS 16.0 and Statistica 7.0.

Results and discussion

Bioremediation is a potentially important option for restoring of oil-polluted environments by exploiting the degradation capabilities of the plant and microbes. However, before starting a phytoremediation project, determining the toxicity of the diesel toward the plant and microbes is essential. It is also important to select microorganisms with potential to grow on petroleum contaminants. Germination percentage of chickpea seeds in LCD was 66%. Microorganisms with potential to degrade diesel were screened from various hydrocarbon polluted soils obtained from petrol pumps and oil refineries. T. ressei was selected from hydrocarbon polluted soil sample obtained from Jamnagar oil refinery (latitude 22°30′N and longitude 70°06′E), Jamnagar a coastal town of Gujarat in India, among 500 microbes screened due to its ability to tolerate 2.5% diesel (data not provided).

Trichoderma ressei had stimulatory effect on the plant growth parameters as compared with un-inoculated control chickpea plant. Root length, shoot length, plant dry weight and chlorophyll content enhanced 128, 31, 46, 79%, respectively, as compared over the un-inoculated control (Table 1). In general, chickpea plants tolerated diesel well at HCD though albeit less efficiently as compared with at LCD (Table 1). At LCD in the presence of T. ressei chickpea root length, shoot length, plant dry weight and chlorophyll content was maximum indicating that at this concentration of diesel chickpea plants could grow very well and amendment of the soil with T. ressei resulted into synergistic effect (Table 1).

Table 1 Effect of Trichoderma ressei on chickpea plant growth promotion and rhizosphere microflora in presence and absence of diesel
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Enumeration of cultivable heterogeneous microbial population demonstrated variable effects as, upon addition of either diesel or, T. ressei impacted native microbial population in soil (Table 1). Effect on heterogeneous bacterial population was most evident at HCD resulted in reduction of 4.84 log unit reduction, as compared with LCD which caused reduction of 2.8 log unit reduction (Table 1). Impact of diesel on soil was somewhat lessened in the presence of T. ressei as reduction in the heterogeneous bacterial population at LCD was negligible as compared no effect in HCD (Table 1). Population of heterogeneous fungi and Actinomycetes also demonstrated similar trend albeit at different levels (Table 1). Correlations between the organic matter status of soils and soil dehydrogenase activity were analyzed. The results establish that application of diesel improved the organic matter status of soils which was in turn reflected in the higher dehydrogenase activity (Table 1). This could be due to diesel being a good source of hydrocarbon readily available for microbial activity. Generally the enzyme activities in the soil are closely related to the organic matter build-up (Marinari et al. 2006). Dehydrogenase activity is thought to reflect the total range of oxidative activity of soil microflora and consequently may be associated to be a good indicator of microbiological activity (Nannipieri et al. 2008). The dehydrogenase activity was high in diesel amended soil than non-diesel soil control (Table 1). Contrary to non-diesel amended soil in the presence of T. ressei diesel spiked soils had lower organic C content and lower dehydrogenase activities indicating that T. ressei somehow negatively affected resident microbial population along with diesel, which turn may have lowered the organic C content and dehydrogenase activities (Table 1).

Microbial communities have great potential for temporal or spatial change, and thus represent a powerful tool for understanding community dynamics in both basic and applied ecological contexts. The small size and rapid growth of microorganisms allow for complex community interactions to be studied much more readily than with plants or animals. Therefore to monitor changes in microbial activity and community composition during bioremediation of a soil sample contaminated with various amounts of diesel oil amended with T. ressei as bioinoculant in the chickpea rhizosphere we utilized a rapid, community-level approach for assessing patterns of sole carbon source utilization by mixed microbial samples using Biolog plate’s shows promise as a means of assessing microbial community structure, which examines the functional capabilities of the microbial population, and the resulting data can be analyzed using multivariate techniques to compare metabolic capability of communities. Biolog plates have found application for the assessment of microbial metabolic diversity to differentiate microbial communities from diverse habitats representing freshwater, sea water, coastal lagoon, soil, rhizosphere, phyllosphere, groundwater, activated sludge reactors, and compost (Garland 1996; Gomez et al. 2000; Nautiyal et al. 2008; Sala et al. 2008). Average well color development (AWCD) based on substrate utilization pattern on Biolog Eco plates by chickpea rhizosphere microflora in presence and absence of T. ressei was elucidated over the period of 7 days (Fig. 1). Data from day 4 was used to ensure that carbon substrates were utilized, thus avoiding false negatives in the analyses based on patterns of colour development on the Biolog plates (Fig. 1). Such information allows examination of the natural variation and diversity of microbial communities. Most importantly, this technique offers the potential to monitor changes in microbial diversity caused by environmental fluctuations, management practices, and pollution. Variable significant differences among treatments were noted for the samples collected using the McIntosh, Shannon, and Simpson indices (Table 1). Significant differences were found among the diversity and evenness indices on effect of diesel on chickpea rhizosphere microflora in presence and absence of T. ressei, based on Tukey’s test (at P = 0.05). Diversity and evenness indices of LCD and LCD + T not showing any significant differences among each other, except McIntosh evenness (Table 1). Principal component analysis of substrate source utilization pattern on Biolog Eco plates by chickpea rhizosphere microflora in presence and absence of T. ressei was determined (Fig. 2). The principal components (PC) score plots describe the characteristics of the samples and help to understand their distribution and clustering. The PCs scores plot (PC-I and PC-II) shows the spatial distribution of the samples. PCA of carbon source utilization pattern on Biolog Eco plates showed carbon source utilization pattern was distributed separately among the six treatments, i.e., control (C); control + T. ressei (C + T); LCD; LCD + T; HCD and HCD + T in the chickpea rhizosphere microflora, at 82.11 and 8.31% on factor 1 and 2 axis. Thus there was distinct resolution of soil microbial communities in the presence of either diesel or, T. ressei (Fig. 2). PCA thus indicated that in general addition of either diesel or, T. ressei impacted native microbial community structure in soil. C and C + T were found to be most separated, while HCD and HCD + T were closely clustured, as compared with LCD and LCD + T (Fig. 2).

Fig. 1
figure 1

Average well color development (AWCD) based on substrate utilization pattern on Biolog Eco plates by chickpea rhizosphere microflora in control (C); control + Trichoderma ressei (C + T); 20 g diesel/kg soil (LCD); LCD + T; 80 g diesel/kg soil (HCD) and HCD + T in the chickpea rhizosphere microflora, respectively. The rate of utilization is indicated by the reduction of tetrazolium, a redox indicator dye, which changes from colorless to purple. Data were recorded for day 1–7 at 590 nm. Microbial activity in each microplate, expressed as AWCD was determined as described by Garland (3)

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Fig. 2
figure 2

Principal component analysis (PCA) of carbon source utilization pattern on Biolog Eco plates (Biolog, Inc., Hayward, CA, USA) of control (C); control + Trichoderma ressei (C + T); 20 g diesel/kg soil (LCD); LCD + T; 80 g diesel/kg soil (HCD) and HCD + T in the chickpea rhizosphere microflora, respectively. Biolog Eco plates were used to determine the effect of diesel on the microbial diversity Rhizosphere of Chickpea grown in Diesel Fuel-Spiked Soil amended with Trichoderma ressei. Data on Biolog Eco plates were recorded every 24 h at 590 nm with an automated microplate reader (BioTek Instruments Inc., USA). At 4th day PCA was performed on blank subtracted data divided by the average well color development (AWCD) as described by Garland and Mills (4). The plotted data are averages of three independent experiments. PCA was performed using STATSTICA 7.0

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In conclusion, our results showed that in soil addition of either diesel or, T. ressei impacted microbial population and native microbial community structure in soil. Characteristics of the dynamics in microbial communities complemented well with organic matter status of soils and dehydrogenase activity. The technique highlighted the usefulness of this parameter for ecological indication of land use change in diesel contaminated ecosystems. Thus application of diesel improved the organic matter status of soils which was in turn reflected in the higher dehydrogenase activity, due to diesel being a good source of hydrocarbon readily available for microbial activity. To the best of our knowledge it is the first report which may constitute a very useful evidence for usage of diesel from C-sequestering point view and will be very useful for future work, especially in view of the characteristics of the dynamics in microbial communities which complemented well with organic matter status of soils and dehydrogenase activity. We envisage that better understanding of soil microbial diversity and microbial efficiency of diesel contaminated soil will be of importance to predict the fate of possible diesel biodegradation under field conditions (Table 2).

Table 2 Effect of diesel on diversity and evenness indices based on substrate utilization pattern by chickpea rhizosphere microflora in presence and absence of Trichoderma ressei
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