A Short Term Study on Toxic Effects of Distillery Sludge Amendment on Microbiological and Enzymatic Properties of Agricultural Soil in a Tropical City

Distillery sludge application in degraded soils is amongst the most important economical resources for the soil fertility amelioration through improvement in soil water-holding capacity, texture, structure, nutrients retention, roots penetration, and reduction in soil acidity [1-3]. Presently due to the ever increasing number of sugar mills and distillery units in the country, it is now become mandatory to use the distillery sludge as a fertilizer to enhance the fertility levels of the soil. However, its application in soil also results in environmental problems [4] because apart from organic content and nutrients, sludge also includes toxic compounds, heavy metals, coloured compounds, dissolved inorganic salts, chlorinated lignin, and phenolic derivatives [5]. Thus, soil microorganisms may be affected via variation in soil temperature, pH, nutrient status, heavy metals, oxygen level and which in turn can affect the ecological processes linked with nutrients cycling [6]. Soil microbial activity, often linked with fertility levels, has been found to be indicated by microbial biomass, microbial respiration, hydrolysis of fluorescein diacetate (FDA), and activity of phosphatase and urease [7-9]. Microbial biomass is the 1-5% active fraction of organic matter of the soil, related with the soil fertility and is highly susceptible to soil pollution than the bulk organic matter. It acts as a reservoir of nutrients and gradually releases it into soil with passage of time which influences soil fertility levels. Soil respiration determines the effect of sludge amendment on soil microbial activity terms of carbon dioxide evolution. In this study, we have chosen enzymatic activities representative of main steps of soil biogeochemical nutrient cycles, i.e. C (FDA hydrolytic activity), N (urease), P (phosphatases), and microbial biomass (Dehydrogenase and FDA hydrolytic activity). Fluorescein diacetate has been used to determine amounts active microorganism. Hydrolysation product of FDA by a number of soil enzymes, such as proteases, lipases, and esterases is fluorescein, which can be easily visualized to estimate microbial activity. Urease catalyzes the hydrolysis of urea and amides to carbon dioxide and ammonia and helps in nitrogen and carbon cycling. Phosphatase is involved in phosphorus mineralization in soils through attack on phosphomonoesterase bond presents in soil organic matter thus increasing the phosphorus availability to microorganisms and plants. Dehydrogenase is a good indicator of metabolic activity soil microorganisms as it is not active independently of the parent microbial cell as extracellular enzymes in soil [10]. Short term evaluation of distillery effluent application in crop fields showed positive effect on crops growth [11-14] but long-term in-depth understanding of effect of distillery sludge is lacking on soil microbiological properties responsible for sustainability of soil. The present study, carried out in Indian tropical agro ecosystems, aims to evaluate the effect of varying amount of distillery sludge on soil microbiological characteristics including major soil microbial groups, microbial respiration, microbial biomass carbon and nitrogen, FDA hydrolysis, phosphate activity and some other soil fertility indices.

Experimental design, sampling and analysis

The study was performed at the agricultural farm at the Narang distillery at Nawabganj (26°52’ N, 82°08’ E and 98 m above the mean sea level). Experiment was designed in randomized block design with four replicate plots of 5 m×5 m size for various doses of sludge amendment. Likewise four un-amended plots were also established as control. The sludge was collected from sludge bed of ETP of distillery, sun dried in the form of cakes and placed in clean sterile polythene bags. Before application, distillery sludge was dehydrated and applied as single dose at a rate of 10, 50, 100 and 150 t ha^-1 (on dry weight basis) in January 2009. Composite soil samples were collected from A-horizon (0–20 cm soil depth) of the plot without any crop at different time intervals from the experimental field. The experimental soil is an inceptisol with a pale brown colour, and sandy loam texture. The soil consisted of 9.5% clay, 10.5% silt, 16.5% coarse silt, 36.7% fine sand, 21.0% coarse sand, and 5.8% humic acids. Ten soil samples were collected and composited into one sample then packed in sterile polythene bags and were stored at 4°C in the dark and maintained at 50% water holding capacity then it was sieved through a 2mm-pore size sieve before use. The physico-chemical properties of sludge, amended and un-amended soil including organic matter, cation exchange capacity were estimated using standard methods [15,16]. Moisture content was determined by wet oxidation method. pH of sludge, un-amended and different sludge-amended soil (1:5 soil/sludge water suspensions) was determined by using digital pH meter. The electrical conductivity (EC) of was measured (1:5 soil/sludge water suspensions) with the help of conductivity meters. Samples were analyzed for total Nitrogen, Phosphorus and Potassium the extractable heavy metal concentrations in soil samples were measured by atomic absorption spectrometry after extraction with aquaregia.

Incubation and estimation of microbial respiration

Basal respiration was determined by the carbon dioxide released in the process of microbial respiration during 10 days of incubation after sludge application. Homogenized samples of fresh moist soil of 500 g (oven-dry basis at 105°C, 24 h), each for control and soil sludge mixtures, were placed into one l capacity jars, sealed properly and further incubated for at 25°C. The incubation of moist soil of each treatment in sealed jars was conducted for 180 days in a growth chamber at constant temperature (25±2°C); the humidity was adjusted to 50-60% of water holding capacity (WHC) and maintained at a constant level throughout the experiment by distilled water filled vial. Two 20 ml beakers were placed on top of the soil. One beaker contained 10 ml 0.5 M NaOH to absorb CO₂ and the other contained 5 ml 2% H₃BO₃ to absorb NH₃. The NaOH solution was changed after every 15 days. The H₃BO₃ solution contained phenolphthalein diluted into 100 mL ethanol (60%, v/v) as an indicator and was titrated once at the end of the incubation. Cumulated microbial respiration of the soils and soil-sludge mixtures were estimated by measuring CO₂ evolution by alkali absorption of CO₂ evolved periodically after each 15 days followed by titrating the residual NaOH with a solution of 0.1 M HCl after precipitating the carbonate with 1 mL of 1.5 M BaCl₂ solution using phenolphthalein as indicator. The data were expressed as mg CO₂-C 100 per gm of soil (Table 1) [17,18].

Table 1: Physicochemical properties of soil, distillery sludge, and sludge amended soil (values represent mean n=3 ±SE). All values presented in mg kg^-1 except electrical conductivity (mScm^-1) and pH.

Soil microbial biomass carbon

Chloroform fumigation-extraction method [19] for the estimation microbial biomass carbon in soil, is considered as a representative method to estimate the whole soil functional entity [20-22]. 25 g each of fresh moist soil and soil-sludge mixture were fumigated with alcohol-free CHCl₃ for 24 h at 25°C in a closed vessel. The organic carbon content of fumigated and control soil and soil sludge mixture was extracted with 0.5 M K₂SO₄ and further estimated by treatment with a mixture of 0.5 M sodium dichromate and sulfuric acid. An extraction efficiency coefficient of 0.45 was used to convert organic C to MBC [23]. Microbial biomass C was calculated as E₂/k_EC, where EC is organic C extracted from fumigated soils - organic C extracted from non-fumigated soils and k_EC 0.45 [24-26].

Number of culturable microorganisms

Microbial populations in both amended and unamended fresh soil samples of 1 g each (in triplicate) were enumerated following standard serial dilution plating technique on selective media using one-fourth strength Ringers solution [27] and expressed as CFU g-1 dry soil. Culturally viable bacteria, fungi and actinomycetes were counted on nutrient glucose agar, Martin’s Rose Bengal Agar (amended with 30 mg/l streptomycin sulphate), and Kenknight’s Agar (amended with 0.05 g/l cyclohexamide), respectively [28]. Plates inoculated with 0.1 ml soil suspension cultured for 4, 7 and 10 days for heterotrophic bacteria, fungi and actinomycetes, respectively, at 28°C [29]. Nitrogen fixing bacteria enumeration in soil was based on the most probable number technique, using a semi-solid nitrogen-free combined carbon medium [30]. Microbial growth associated with acetylene reduction (indicating nitrogen-fixing bacteria) was assessed with the help of gas chromatograph Varian 3800GC (Varian Analytical Instruments, USA).

Enzyme activity

FDA activity: FDA activity (representing soil organic matter decomposition) was measured by incubating soil and soil-sludge mixtures (both 1.5 g) with 9.9 ml sodium phosphate buffer [8]. Hydrolysis, started at addition of 0.1 ml FDA solution (1 mg ml^-1), was stopped after 1 hour of incubation at 25°C by adding 10 ml of acetone and absorbance of supernatant filtrate (by Whatman No. 40 filter paper) was measured at 490 nm. The concentration of the released fluorescein was calculated by pre-drawn standard curve and expressed as μg FDA g^-1 h^-1.

Urease: 0.5 mL toluene and 12 mL phosphate buffer mixed with 3g sieved soil and soil sludge mixtures and incubated for 15 min at 37°C in a BOD incubator. After mixing with 3 ml of 10 % urea solution it was again incubated for 1 hr at 37°C and 15 mL of a KCl 2 M solution containing 5 mg phenyl mercury acetate were added. The sample was agitated for 5 min and then filtered through a Whatman no.1 filter paper. 0.2 g magnesium oxide previously heated at 500-600 8°C for 1 hr was added to 10 microlitres of the filtrate to remove carbonates. The filtrate was added to 5 mL boric acid solution, with methyl red and bromocresol green as indicators and then titrated with a H₂SO₄ 0.001 M standard solutions. A blank was prepared for each treatment, and the same procedure utilized for the samples was adopted, with the only difference that the urea solution was added after the KCl and phenylmercury acetate solution. Urease activity was expressed as μg N-NH₄ ⁺g^-1.produced in 1 h of incubation [31].

Phosphatase: 5 g of soil sample, 0.25 ml of toluene, 1ml of 10 mM p-nitrophenyl phosphate and 10 ml of distilled water were mixed and after 1hr incubation 5 ml of 0.5 M CaCl₂ and 20 ml of 0.5 M NaOH were added. The content was filtered using Whatman No.42 filter paper and volume made up to 50 ml with distilled water. The colour intensity was read at 420 nm. The concentration of phosphatase was obtained from a standard graph and the enzyme activity was expressed as μmol PNF g^-1 h^-1 [32,33].

Dehydrogenase: A 20 g of soil sample was taken in a boiling tube. To this 1 ml of 3% 2, 3, 5-Tri phenyl tetrazolium chloride (TTC) was added. Then 1 ml of 1 % glucose and 2.5 ml of distilled water was added and incubated for24 hrs at 37°C. After that 10 ml of methanol were added and incubated for another 5 hrs. The content was filtered and the samples were washed thoroughly with methanol. The red colour developed was read at 485 nm. The concentration of dehydrogenase in the sample was obtained from the standard graph using triphenyl farmazane. Results were expressed in μg TPF g^-1 day^-1 [33,34].

Soils and sludge characteristics

Physico-chemical analysis of sludge was done in triplicate sets of samples showed that all the collected samples have high pH, EC, BOD, COD, TSS, TDS, phenolics and metals (Cu, Cd ,Cr, Zn, Fe, Ni, Mn, Pb etc) as shown in. High concentrations of nitrogen, phenol, chloride and heavy metals were detected in distillery sludge versus the agricultural soil .The values of pH (9.2) and EC (3.25 mScm⁻¹) were also higher in sludge than the soil where the pH and EC were 6.8 and 1.55 mScm⁻¹respectively. High values of pH and EC in the sludge may be due to the presence of high concentrations of soluble salt. High concentrations of heavy metals and salts in sludge are due to the condensation process, which takes place during sugar manufacturing and alcohol production. The effect of distillery sludge treatment on some soil chemical properties is also shown in the same which shows that certain properties increase from the control soil after amendment.

Respiration

The cumulative CO₂ for control soil, sludge and for different soil sludge after 180 days of different soil sludge amendments was calculated. The microbial respiration of the sludge was many times higher than observed in soil. The addition of sludge increased the CO₂ production of soils initially, increasing almost ten times the respiration activity when 150t ha^-1 was applied, in respect to the unamended soils and then declined (Figure 1). This increment is associated with large amount of easily available soluble organic matter [3], nitrogen, phosphorous and other nutrient sources of the sludge that would have triggered microbial activity causing high rate of microbial-mineralization in the soil-mix [35]. The moistening of soil through addition of sludge plays a major role in increase in respiration which reactivates the growth and activity of microorganisms. The decline in CO, evolution in the later stage is caused by the exhaustion of one or more essential nutrients and the accumulation of toxic metabolites during incubation at higher doses of sludge application [36].

Figure 1: Changes in Microbial biomass carbon and Microbial basal respiration in distillery sludge amended soil at rates of 10, 25, 50, 100, 150 t ha-1 for varying time of incubation (the mean values are presented, n=3).

Microbial biomass C

Soil microbial biomass and biological activities is the indicator for changes in soil resulting from different stresses in soil ecosystems [37]. It was found from the experiment that the microbial biomass carbon in soil increased with the increment of sludge application and higher level (mg kg^-1) was obtained after 90 days of incubation with the application of 150 t ha^-1 of sludge (Figure 1). After 120 days of incubation, the MBC decreased in amended soil. It was seen that that application of water alone increases MBC. However, the values of MBC in amended soils were always higher than that of unamended control soil. All the incubation experiment is carried in controlled condition so that effect of seasonal change and other environmental factor in microbial biomass can be removed.

Microbial population

Quantitative analysis of soil microbial populations show variations in the populations of different groups of microflora in the soils following amendment with distillery sludge (Figure 2) The results show a marked decrease in total culture numbers of the different microbial groups for the highly contaminated soil samples. Aerobic heterotrophic, nitrifying and denitrifying bacteria seem to be more sensitive to high dose of distillery sludge contamination than the other microbial groups under evaluation. A difference in the viable counts of fungal and actinomycetes were also significant; however, these two microbial groups seemed to be less sensitive to the presence to the amendment.

Figure 2: Changes in microbial population (CFU per gram of dry soil) sample following amendment with different amount of distillery sludge for varying length of time (all values are mean n=3, and SE bars are shown).

FDA activity

The FDA activity in soil increased with the increment of sludge application, and the higher level was obtained with the application of 150 t ha^-1 of sludge, after 60 days of incubation (Figure 3). Results of FDA hydrolysis obtained with soil throughout the all incubation period (0-180 days), the FDA hydrolysis in sludge-amended soil was significantly different (P<0.05) compared to unamended. After 90 days of incubation, the FDA values decreased in both amended and unamended soil, however, the changes in values of FDA obtained in unamended soil was not very much significant. The increase of FDA hydrolysis indicates increment in the activity of the microorganisms due to organic matter contribution, macro and micronutrients, and pH increment by the sludge application. All these elements are essential for microbial growth and soil activity.

Figure 3: Changes in different enzymes activities in soil amended with varying amount of distillery sludge for varying days of incubation (the mean values are presented, n=3).

Urease activity

The mean urease activity was highest at the 150 t/ha application of distillery sludge i.e. while control shows the lowest activity (Figure 3). Enzyme activity changes both according to the level of application and incubation period during the experiment it was also seen that after application the soil show rapid increase in urease activity during incubation period when no additional amendment is applied. A slight decrease in urease activity is seen at 100 t/ha application of the sludge. Urease activity was expressed as mg N–NH+4 produced in 1 h of incubation.

Dehydrogenase activity

Dehydrogenase activity increased significantly at 150 tha^-1 amendment on the 10^th day of incubation (Figure 3). The activity increased within the period of incubation as reflected in the mean values within the days of incubation respectively. Dehydrogenase is an important enzyme for the oxidative processes in the soil which increases gradually with the rate of sludge application and its activity is very much dependent on the organic matter supply through distillery sludge. Our results are in agreement with [38,39] who also considered the activity of dehydrogenase, microbial biomass, soil respiration, and counts of Nfixing bacteria, etc. as sensitive bio-indicators of soil quality μgTPF/g.

Phosphatase activity

The phosphatase activity in distillery sludge treated soil is maximum when 150 t/ha sludge is applied and it further increases with the increase of the duration of incubation (Figure 3). It was maximum i.e. at the 180^th day for the 150t/ha sludge application. Phosphatase recorded the maximum activity of 140.6 g kg hr that might have been favored by the copious quantity of phosphate present in the effluent. Sludge addition in experimental soil increases the plant-available phosphorus content and decreases acidity of the soils and consequently increases available P [40]. An increase of phosphorus availability in soil have been well correlated with the increase of phosphatase activity, being an adequate indicator of microbial activity and of modifications occurred in the soil due to sewage sludge application [9,33,41]. The increases the phosphatase activity is shown throughout incubation time (0-180 days), being significantly different (P<0.05) compared to unamended soils. The maximum activity was observed at 60 days of sludge application, and then the phosphatase activity decreased. experimental soil the levels of this enzymatic activity was 5 times higher after 60 days of incubation when 150 t ha^-1 of sludge was applied, compared to unamended soil. The highest acid phosphatase activity observed in sludge-amended soils is similar to the results obtained for microbial biomass carbon and FDA hydrolysis, indicating that the sludge addition contributes to increase the overall microbial activity in this soil.

It is observed that distillery sludge amendment influences physical, chemical and microbiological properties of the agricultural soil [8] which is in turn vital for the nutrient turnover and productivity [20,22,40]. Generally it is expected that the microbial activity in soil would always increase with the application of organic matter as distillery sludge, but, on higher doses negative effect by simultaneous enrichment of organic pollutants and heavy metal salts causes reduction in microbial biomass, activity and microbial populations of the soil. Experimental soils increased in organic matter, electrical conductivity, soluble heavy metals and toxic organic contaminants up to toxic level. The buffering capacity of the soil to degrade the pollutants is also not very effective at such a large quantity of toxic chemical addition [42]. The toxic effects of heavy metals and toxic organic substances present in distillery sludge on microbes are also determined by their solubility which is influenced its pH. This work showed that microbiological properties (MBC, microbial respiration, enzyme activity) increased as increasing rates of distillery sludge addition soils. Here the amount 50t ha^-1 to100 t ha^-1exhibited increasing tendency in terms of biological properties from 60 days to 120 days after application, thereafter it became lower at 180 days.

The quantitative analysis soil samples collected at different time period after different amount of application of distillery sludge showed continuous increasing counts of bacteria, fungi, and actinomycetes initially which were according to soil organic C and nutrient status when the concentration of the pollutants in the soil are below the toxic level then the number decreases drastically. At low doses it showed microbial population of reduced diversity but of greater number, biomass and activity, but at higher doses it causes decrease in number of microorganism, corresponding enzymatic activities and other microbiological properties. Aerobic heterotrophic and nitrogen fixing bacteria seem to be more sensitive to the toxic effects of distillery sludge than the other microbial groups under evaluation, undergoing a decrease in population size on higher doses. Differences in the viable count of fungal and actinomycetes were also significant; however, these two microbial groups seemed to be less sensitive to higher doses of sludge application in the soil.

The addition of sludge also increased the CO₂ production and microbial biomass carbon of soils initially, increasing almost ten times when 150 tha^-1 distillery sludge was applied, in respect to the unamended soils and then declined during the incubation period. Highest level was obtained after 90 days for MBC and 120 days for respiration of incubation with the application of both 150 t ha^-1 and 100 t ha^-1 of sludge then they decreased.

Enzyme activity changes both according to the level of application and incubation period during the experiment. The FDA hydrolysis activity in soil increased with the increment of sludge application, and the higher level was obtained with the application of 150 t ha^-1 of sludge, after 60 days of incubation. After 90 days of incubation, the FDA values decreased, however, the changes in values of FDA obtained in unamended soil was not very much significant. Dehydrogenase activity increases up to 90^th day of incubation. The activity increased many times within the period of incubation as reflected in the mean values. The phosphatase activity in distillery sludge treated soil is maximum when 150 t/ha sludge is applied but it further decreases with the increase of the duration of incubation. It was maximum i.e. at the 60th day for the 150t/ha sludge application. There was a rapid increase in urease activity initially in all the amendments and in the last phase of incubation there is a decrease in activity.

Distillery sludge amendment increased organic matter and electrical conductivity in the experimental soils and at high doses the amounts of soluble heavy metals and toxic organic contaminants were also higher up to toxic level. This work showed that microbiological properties (MBC, microbial respiration, enzyme activity) increased as increasing rates of distillery sludge addition in agricultural field soil in controlled conditions. Here the amount 50t ha^-1 to 100 t ha^-1 exhibited increasing tendency in terms of biological properties from 60 days to 120 days after application, thereafter it became lower at 180 days. 150 tha^-1 distillery sludge shows negative effect on these properties comparatively very early. At the higher concentration of distillery sludge, decreasing tendency of soil fertility indicators observed becomes faster hence, we recommend the use of distillery sludge as improver of biological properties of soils if used in low concentration previously verifying its level of toxicity and stability to avoid potential hazardous effects over the soil microorganism. To minimize the shown environmental risks an adequate sludge management is proposed.

The authors are thankful to the University Grant Commission, New Delhi for their financial assistance and the Centre for Environmental Science & Technology, BHU, Varanasi, for technical help.