INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 2, No 2, Copyright 2010 All rights reserved Integrated Publishing Association

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1 INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 2, No 2, 2011 Copyright 2010 All rights reserved Integrated Publishing Association Research article ISSN Biodiesel Spentwash: Characterization, Amelioration and its effect on Seed Germination, Seedling Growth and Biochemical Parameters of French bean (Phaseolus vulgaris L.) Mohana.V.S 1, Balakrishna Gowda 2, Pramila.C.K 3, Prasanna.K.T Ph. D scholar, Division of Environmental Sciences, IARI, New Delhi Professor, Department of Forestry and Environmental Sciences, UAS, GKVK, Bangalore Research Associate, Department of Seed Science and technology, UAS, GKVK, Bangalore - 65 mohanfcp@gmail.com doi: /ijes ABSTRACT A laboratory study was conducted to characterize the biodiesel spentwash samples, amelioration by physical and chemical methods to improve the quality and to know the effects of treated and untreated biodiesel spentwash on seed germination, seedling growth, dry matter production and biochemical parameters of French bean (Phaseolus vulgaris L). The biodiesel spentwash samples were collected from biodiesel production unit of UAS, Bangalore and used for the study. Different ameliorative methods were followed to treat the spentwash samples which include sand filtration, chemical coagulation etc., the treated and untreated spentwash samples were used for the germination experiments. Treated spentwash samples showed very good response in seed germination and other growth parameters, whereas the untreated (Raw Biodiesel Spentwash) samples showed inhibitory effects. The study suggests that biodiesel spentwash can be safely used for irrigation after proper treatments. Key words: 1. Biodiesel spentwash, 2. Dry matter production, 3. Biodiesel production unit, 4. Chlorophyll content 5. Carotenoides. 1. Introduction Water is one of the most important precious resource found on the earth. The water resources are most often affected by anthropogenic activities and also from industries. Pollution caused by human beings and industries is a serious concern throughout the world. Growth of population, massive urbanization, rapid rate of industrialization and modern techniques in agriculture have accelerated the water pollution and led to gradual deterioration of its quality. Due to continuous disposal of waste water into the water bodies, surface water quality throughout the country has deteriorated because of the mixing of various chemical pollutants of the effluent with water. The surface water quality has declined to such a level at which it will not be useful for any purpose. The use of industrial liquid effluent for agricultural irrigation has been in vogue in many countries of the world. This effluent contains various micro nutrients essential for growth of crop plant. However, many industrial wastes may have harmful effects and may cause soil fatigue. Biodiesel spentwash is a wastewater discharged from the biodiesel production units during the process of biodiesel production, where biodiesel was washed with water to remove the Received on September 2011 Published on November

2 suspended matter and catalysts present in it. About 2.5 to 3 liters of wastewater (spentwash) was produced for production of a liter of biodiesel (Hanny and Shizuka, 2007). The biodiesel spentwash was dark yellowish cream in color, highly turbid, pungent smelling and has high organic load as seeds are used as a chief raw material in the production, with high levels of chemical oxygen demand, biological oxygen demand, oil, high suspended and dissolved solids, high amount of sodium, nitrogen, phosphorus and potassium content (Suehera et al., 2005). To recycle nutrients through land application of biodiesel spentwash requires the use of crops capable of utilization of these nutrients. Industrial effluents rich in organic matter and plant nutrients are finding agricultural use as cheaper way of disposal. Since most of the wastewater is being discharged into the surrounding water bodies, which disturbs the ecological balance and deteriorates the water quality, therefore, present study has been carried out to study the impact of biodiesel spentwash (treated and untreated) on seed germination, seedling growth, dry matter production and biochemical parameters of French bean variety KMP101-Tanu. 2. Materials and Methods 2.1 Collection of samples The spentwash samples derived from Karanj (Pongamia pinnata L.) biodiesel were collected from Biodiesel Production Unit (BPU), GKVK, UAS, Bengaluru, Karnataka, India, in plastic containers from the point of disposal brought to the laboratory and analyzed for various physico-chemical properties as per the methods prescribed by APHA (American Public Health Association, 1998) handbook. 2.2 Amelioration of biodiesel spentwash The biodiesel spentwash samples were treated by using different ameliorative techniques like filtration through sand filters at different time intervals (Rodgers et al., 2005) and chemical coagulation with different chemicals (Mohana et al., 2011a). The design of the experiment was shown below Experimental design Treatment symbol Control T 1 T 2 T 3 T 4 T 5 T 6 T 7 S 1 S 2 S 3 Treatment details Tap water Raw biodiesel spentwash Calcium hydroxide at 75 mg/l Calcium hydroxide at 125 mg/l Ferric chloride at 100 mg/l Ferric chloride at 200 mg/l Potash alum at 50 mg/l Potash alum at 100 mg/l Sand filtrate at 10 minutes Sand filtrate at 30 minutes Sand filtrate at 60 minutes 1040

3 The treated and untreated biodiesel spentwash samples were used for the germination and other growth related studies. 2.3 Germination studies French bean seeds were surface sterilized with 0.1% mercuric chloride (HgCl 2 ) and washed with distilled water. Twenty five seeds of french bean were placed equi-spacially in sterilized petriplates, lined with filter paper soaked with spentwash from different treatments and distilled water. These petriplates were irrigated with spentwash samples from different treatments uniformly. Number of seed germination was counted on 10 th day and total germination percentage was calculated. Data were taken from three replicates of seedling on 10 th day old seedlings. Tenth day old seedlings were separated into root and shoot and were used for estimation of dry matter production, chlorophyll (Arnon, 1949), carotenoid (Kirk and Allen, 1965), sugars (Nelson, 1994), protein (Lowry et al 1951), starch (Summer and Somers, 1949) and amino acid content (Moore and Stein, 1948). 2.4 Statistical analysis Fisher s method of analysis of variance was employed for the analysis and interpretation of the data as given by Gomez and Gomez (1976). The level of significance used in F test was P=0.05 and the CD values were calculated. Table 1: Physico-chemical analysis of biodiesel spentwash Sl. No. Parameters Raw Biodiesel Spentwash General parameters 1 Colour Dark yellow with brownish residues and turbid ISI tolerance limits for irrigation 2 Odour Pungent Odourless 3 ph Electrical Conductivity 1.20 < Oil content Total Suspended Solids Total Dissolved Solids Biological Oxygen Demand Chemical Oxygen Demand Chemical parameters 10 Nitrogen Potassium Phosphorus Sodium Clear 1041

4 All parameters were expressed in mg/l except temperature, ph, EC, colour and odour. 3. Results and discussions The physico-chemical properties of biodiesel spentwash were given in Table 1. The spentwash is dark yellow creamish in colour, with brownish residues and having pungent smell which is slightly irritating in nature. Spentwash is highly alkaline, consist of high amounts of oxygen demanding waste, high amounts of dissolved and suspended solids. Spentwash also consist of high amounts of plant nutrients. Similar findings were also reported by Suehera et al., (2005). Table 2: Effect of different ameliorative techniques on properties of biodiesel spentwash T 1 : Raw biodiesel spentwash T 2 : Calcium hydroxide at 75 mg/l T 3 : Calcium hydroxide at 125 mg/l T 4 : Ferric chloride at 100 mg/l T 5 : Ferric chloride at 200 mg/l T 6 : Potash alum at 50 mg/l T 7 : Potash alum at 100 mg/l S 1 : Raw biodiesel spentwash S 2 : Sand filtrate at 10 minutes The different ameliorative techniques such as sand filtration and chemical coagulation were improved the quality of spentwash by reducing the pollutants present in it. The results were S 3 : Sand filtrate at 30 minutes S 4 : Sand filtrate at 60 minutes : Significant at 5% shown in the table 2. The maximum ameliorating effect was recorded in treatment S 3. This could be due to salts and other soluble materials present in the spentwash that adhered to the minute sand particles and thus reducing the concentration of salts. The microorganisms 1042

5 present on the surface of the sand filter could have decomposed the organic matter in the spentwash and reduced the organic waste from the spentwash. Similar findings were reported by Mohana, et al., (2011a), where the authors treated the spentwash samples by using different physical filters. 3.1 Effect of treated and untreated spentwash on germination and growth parameters of French bean The treated spentwash samples showed promoting effect on seed germination, seedling growth, and dry matter production in French bean. Whereas untreated samples showed inhibitory effect on seed germination and other growth parameters. The results were showed in table 3 and figure 1. Table 3: Effect of treated and untreated biodiesel spentwash on seed germination, seedling growth and biomass accumulation of French bean (Phaseolus vulgaris L) Treatments Germination (%) Seedling growth (cm) Fresh weight (gm/ plant) Dry weight (gm/ plant) Control: Tap water T 1 : Raw biodiesel spentwash T 2 : Calcium 75 mg/l T 3 : Calcium 125 mg/l T 4 : Ferric chloride mg/l T 5 : Ferric mg/l T 6 : Potash 50 mg/l T 7 : Potash 100 mg/l S 1 : Sand 10 minutes S 2 : Sand 30 minutes S 3 : Sand 60 minutes F Value CV (%) S. Em ± CD at 5 % NOTE: Significant at 5% The germination, seedling growth and dry matter production of French bean are significantly differed in different treatments. The maximum promoting effect was recorded at treatment S 3 (Sand minutes). The least was recorded in raw effluent (T 1 ). The maximum promoting effect was observed in treated samples might be due to reduction in pollutants in the filters. Germination percentage, seedling growth and dry matter production were inhibited in raw spentwash. It might be due to osmotic pressure caused due to high dose (Dhanam, 2009). Osmotic pressure of the spentwash at higher concentrations of total salts making imbibition 1043

6 Biodiesel Spentwash: Characterization, Amelioration and its Effect on Seed Germination, Seedling Growth and was more difficult and retarded the germination. Presence of high amounts of sodium makes the spentwash highly alkaline, which also inhibits the germination and seedling growth of french bean. The results were supported by the findings of Prabhakar et al. (2004) where, authors investigated the effect of fertilizer factory effluents on seed germination, seedling growth and chlorophyll content of gram seeds and reported that lower concentrations of effluents promote the seed germination and seedling growth whereas, it was inhibited at higher doses of effluents. Table 4: Effect of treated and untreated biodiesel spentwash on biochemical parameters of French bean (Phaseolus vulgaris L) Carotinodes (mg/g fw) Proteins Starch Total Sugars Amino acids Treatments Chlorophyll a (mg/g fw) Chlorophyll b (mg/g fw) Total chlorophyll (mg/g fw) Root Shoot Root Shoot Root Shoot Root Shoot Control: Tap water T 1: Raw biodiesel spentwash T 2: Calcium 75 mg/l T 3: Calcium 125 mg/l T 4: Ferric mg/l T 5: Ferric 200 mg/l T 6: Potash 50 mg/l T 7: Potash 100 mg/l S 1: Sand 10 minutes 1044

7 Biodiesel Spentwash: Characterization, Amelioration and its Effect on Seed Germination, Seedling Growth and S 2: Sand 30 minutes S 3: Sand 60 minutes F Value CV (%) S. Em ± CD at 5 % NOTE: Significant at 5% Biochemical parameters viz., chlorophyll, carotenoids, total sugars, starch, protein and amino acid contents increased at lower concentrations, were maximum at treatment S3 (Sand minutes) [Table 4]. Enhancement of chlorophyll could due to high nutrient uptake, synthesis and translocation probably facilitated by optimum availability of iron and magnesium and also due to reduction in phenol content in the treated biodiesel spentwash While, decrease at raw spentwash. This is supported with previous work on French bean with effect of biodiesel spentwash (Mohana et al., 2011b). Protein, starch, amino acid contents were increased in this study in treated spentwash. The crude protein concentration of corn increased quadratically with increased N fertilizer with dairy effluent application [Leary and Rhem, 1990]. The enhancement of protein content of crop plants might be due to increased rate of amino acid synthesis which may be attributed to the higher rates of both RNA-ase and transaminase activity [Singh, 1991]. The decrease in starch content raw spentwash may be due to the lowered activity of phosphorylase and the increased activity of beta-amylase and invertase with effect of spentwash. Since the higher concentration and untreated spentwash is toxic to the plant growth, it is recommended that only after suitable treatments and proper dilution the biodiesel spentwash be used for irrigation purpose. 4. Conclusions Biodiesel production is one of the most important industry which is gaining importance nowadays. BPU utilizes enormous amount of fresh water during production process and releases huge quantity of polluted water. The physico chemical analysis of wastewater revealed that the wastewater is highly polluted and the quality can be improved by suitable ameliorative techniques. Among the different ameliorative techniques sand filtration was 1045

8 found to be most effective in reducing the pollutants load from the spentwash. The treated wastewater samples showed better results in terms of plant growth, dry matter production and biochemical properties of French bean. By the study it is revealed that biodiesel spentwash can be successfully used for irrigation after suitable treatments and proper dilutions. 5. References 1. Hanny J. B. and Shizuka Hirata (2007), Biodiesel production from crude Jathropa curcus L. seed oil with a high content of free fatty acids. Bioresource tech.,99 (2008), pp Suehara, K. I., Kawamoto, Y., Eiko Fujii., Jiro Kohda., Yasuhisa Nakano. and Takuo Yano (2005), Biological treatment of waste water discharged from biodiesel fuel production plant with alkali-catalyzed transesterification. J. Biosci. and Bioengg., 100(4), pp American Public Health Association (APHA)., (1998),., Standard methods for Examination of Water and Wastewater. 20 th ed. Washington, DC. 4. Rodgers, M., Healy, M. G. and Mulqueen, J (2005), Organic carbon removal and nitrification of high strength wastewaters using stratified sand filters. Waterres., 39(14), pp Mohana V. S., Srinivasa Murthy., Prasanna, K. T and Balakrishna Gowda., (20011a), Characterization of biodiesel spentwash and amelioration through chemical coagulation for irrigation, Environ. Ecol., 29 (3A), pp Arnon, D. I (1945), Copper enzyme is isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol., 24, pp Kirk, J.T.O. and R.L. Allen (1965), Dependence of chloroplast pigments synthesis an protein synthetic effects of acitilione. Biochem. and Biophysical res. Communication, 27, pp Nelson, N (1994), A photometric adaptation of the somogyi s method for the determination of reducing sugar. Anals of Chemistry, 3, pp Lowry, H.O., N.J. Rosenborough, A.L. Farr and R.J. Randall (1951), Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, p Summer, J.B. and G.F. Somers (1949), Laboratory experiments in Biological Chemistry 2 ed. Academic Press, New York, p Moore, S. and W.H. Stein (1948), Photometric method for use in the chromatography of amino acids. Journal of Biological Chemistry, 173, pp Gomez, K. A. and Gomez, A. A., (1976),Statistical procedure for agricultural research II. Edn., John Willey and sons, New York, P Dhanam, S., (2009), Effect of dairy effluent on seed germination, seedling growth and biochemical parameter in French bean. Botanical Res. Int., 2(2), pp

9 14. Mohana, V. S., Nandini, N and Manu, K. J., (2011), Effect of biodiesel spentwash on seed germination seedling growth and biochemical parameter of French bean (Oryza sativa L.) and French bean (Phaseolus vulgaris L.)., Int. J. Res. Chem. Environ., 1(1), pp Prabhakar, P. S., Manisha Mall. and Jaswanth Singh (2004), Impact of fertilizer fertilizer factory effluent on Seed Germination, Seedling growth and Chlorophyll content of Gram (Cicer aeritenum). J. Environ. Biol., 27 (1), pp Leary, M.J. and G.W. Rhem (1990), Nitrogen and sulfur effects on the yield and quality of corn growth for grain and silage. J. Production Agriculture, 3, pp Singh, V.K (1991), Changes induced by phorate in the nitrogen metabolism of black gram. J. Advances in Plant Sci., 4, pp