International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 1(4), 28-32, August (2012) I. Res. J. Biological Sci. International Science Congress Association 28 Pigment Degradation of Higher Plants near Sugar Mill Sarala Thambavani D.1 and SabithaM.A.2*Department of Chemistry, Sri Meenakshi Government Arts College for Women (Autonomous), Madurai-625, INDIA Department of Chemistry, J.A. College for Women (Autonomous), Periyakulam-625 601, INDIA Available online at: www.isca.in Received 25th June 2012, revised 3rd July 2012, accepted 6th July 2012Abstract Sugar industry generate a significant large amount of waste during the manufacture of sugar and contains a high amount of production load particularly in items of suspended solids, organic matters, press-mud, bagasses and air pollution. Air pollutants from sugar mill can directly affect plants via leaves or indirectly via soil acidification. When exposed to air pollutants, most plant experience physiological changes before exhibiting visible damage to leaves. Leaf pigment content can provide valuable insight into the physiological performance of leaves. In the present study, the extraction of chlorophyll and their derivatives were studied in the five tree species such as Ficus benghalensis, Delonix regia, Ficus religiosa, Azadirachta indica and Pongamia pinnata. The study area was polluted with organic pollutants which results in reduced pigment levels in the leaves. The pheophytin a and b were formed greater than hundred percent in Delonix regia Azadirachta indica and Pongamia pinnata. For Ficus benghalensis, pheophytin a was found to be 27.17% and pheophytin b was more than hundred percent. In Ficus religiosa, pheophytin a was formed by 41.09% where as pheophytin b was produced less than the residential area. These results indicate that the degradation of chlorophyll was higher near sugar mill because of the emission of gases. Among the five tree species growing near sugar mill, Ficus religiosa showed minimum decomposition of chlorophyll and it was suggested to grow these species to reduce the load of pollution near sugar mills. Key words: Air pollutants,visible damage,organic pollutants, chlorophyll, degradation, pheophytin a and b. Introduction Sugar industry is one of the most important agro based industries in India and is highly responsible for creating significant impact on rural economy in particular and countries economy in general. Sugar industries rank second amongst mavar agro based industries in India. Sugar industry is seasonal in nature and operates only for 120 to 200 days in a year (early November to April). A significant large amount of waste is generated during the manufacture of sugar and contains a high amount of production load particularly in items of suspended solids, organic matters, press-mud, and bagasses and air pollution1,2,3. Gaseous emissions such as CO, SO and NO were reported both from process and fired equipment from sugar industry. Air pollutants can directly affect plants via leaves or indirectly via soil acidification. When exposed to airborne pollutants, most plants experienced physiological changes before exhibiting visible damage to leaves. Plants provide an enormous leaf area for impingement, absorption and accumulation of air pollutants to reduce the pollutant level in the air environment, with a various extent for different species. Trees provide a large leaf surface onto which particles are deposited and gases are removed. Pollution is removed by nearly all parts of a tree; the soil, roots and vegetative portions of the tree species. Trees respirate and exchange gases through stomata or holes, on their leaves; these gases include those necessary for the tree’s functioning as well as other gaseous air pollutants. Once inside the leaf, gases diffuse into the spaces between the cells of the leaf to be absorbed by water films or chemically altered by plant tissues. Trees also reduce air pollution by intercepting airborne particles and retaining them on the leaf surface, called dry deposition. Some can be absorbed by the leaf surface itself, although most remain on the plant surface10. Previous studies also showed the impact of air pollution on ascorbic acid content11, chlorophyll content12,13, leaf extract pH14,15 and relative water content16. There are many pigments in the higher plants, such as chlorophyll (chl), carotenoid (car), phytochrome, flavinoid, anthocyanin, tannin and so on. Chlorophyll contains two different chemical structures, a and b, while the former contains CH at the position 7 of ring B, the latter contains CHO at the same position. The major absorbance of both chlorophyll a and b is in the blue and red visible light. All carotenoids absorb blue light. Phytochrome transforms its structure between red and far red light. Flavonoids, including anthocyanins and tannins, absorb green and ultra-violet light. These pigments offer an all-oriented network of interception, reflection and protection for plant body, allowing plant to use sunlight and cut off its photodamage. Plant pigments are degraded into small molecules recycling into the nature after completing their mission. Eight steps are involved in the degradation pathway of chlorophyll to pheophorbide17 (figure 1). With acid gases or vapors, the initial action on the chlorophyll pigments is conversion to the corresponding pheophytins. The International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202Vol. 1(4), 28-32, August (2012) I. Res. J. Biological Sci. International Science Congress Association 29 extent of decomposition of chlorophyll or conversion to pheophytin is a measure of the degree of air pollution damage to leaves of plants, and this can be determined readily by spectrochemical methods. Cholorotic injury is the result of a breakdown of the chlorophyll in the leaf and may be either a temporary or permanent condition. The leaf tissue may still exercise its functions, but at a reduced rate. In the transient form, the chlorosis consists of a slight paling of the normal green color which is usually restored within a few days. Where the chlorosis is of the permanent type, the loss in chlorophyll content is evident in the yellow or yellowish'green appearance of the leaves. The gross symptoms of permanent chlorosis are similar in appearance to senescence and may result from the continued absorption of gas and its accumulation as sulfate in the leaf beyond the tolerance limit. When the leaf chlorophyll and other green pigments are decomposed, the leaf assumes a bleached, reddish'brown, brown, or ivory appearance, depending upon the nature of the species. In order to determine the effect of air pollution exposure and damage on the chlorophyll and pheophytin content of living plants, five tree species growing near sugar mill and residential area were selected as the most suitable species for study purposes. Material and MethodsSelection of sampling area and sampling details: The research work was mainly confined near sugar factory. Leaves of five tree species such as Ficus benghalensis, Delonix regia, Ficus religiosa,Azadirachta indica and Pongamia pinnata were collected from polluted area and residential area. The screening and selection of the tree species was partly based on literature survey of similar work and guidelines of Central Pollution Control Board. The five leaf samples were collected at the lower most position of canopy at a height of 6-7ft from the ground surface. Samples were cleaned with distilled water and then refrigerated (22ΊC) under suitable condition for further biochemical analysis. Extraction process: Chlorophyll a, b and Carotenoid: The preweighed samples of five tree species were dissolved in 80% acetone (20ml per each gram) and grained using mortar and pestle and then homogenized using a homogenizer at 1000 rpm for about 5 minutes. Then the samples were filtered using cheese cloth. The extracts obtained were centrifuged at 5000 rpm for about 10 minutes. The supernatants were separated and absorbances were read at 200 – 700 nm on UV spectrophotometer. The experiment was repeated thrice for statistical analysis. The content of chlorophyll a, b and carotenoid were calculated using the equations of Porra et al20and Holm21 respectively. Chl a = 12.25 A663.6 – 2.55 A646.6 (g/ml), Chl b = 20.31 A646.6 – 4.91 A663.6 (g/ml), Car = 4.69 A440.5 -0.267 Chl a+b (g/ml) Porphyrins: The above acetone extract was mixed with equal amount of hexane and kept undisturbed till interface appears. The upper phase contains less polar compounds dissolved in hexane and the lower phase contains more polar compounds dissolved in acetone. The upper and lower fractions were separated and the lower fraction was used to measure the absorbance at 575, 590 and 628 nm. The equation of Khan et al22 was hired to determine the content of protoporphyrin IX (PPIX), magnesium-protoporphyrin IX (MGPP) and protochlorophyllide (Pchlide). PPIX = 196.25 A575 – 46.6 A590 – 58.68 A628 (nmole), MGPP = 61.81 A590 – 23.77 A575 – 3.55 A628 (nmole), Pchlide = 42.59 628 – 34.22 A575 – 7.25 A590 (nmole) Chlorophyllide (Chlide) a and b: The lower acetone fraction was used to measure the absorbance at 667 and 650 nm. The Beer-Lambert law and method of McFeeters23 was used to calculate the content of chlorophyllide a and b. Chlide a = A667/ 74.9 (mM), Chlide b = A650/ 47.2 (mM) Pheophytin (Phe) a and b: The upper hexane fraction was dried with nitrogen and dissolved in 80% acetone. The absorbance were measured at 665.4 and 663.4 nm, which were major absorption peaks of pheophytin a and b respectively. The content of pheophytin a and b were calculated using the equation of Lichtenthaler24. Phe a = 22.42 A665.4 – 6.81 A653.4g/ml), Phe b = 40.17 A653.4 – 18.58 A665.4 (g/ml) Less polar caroteoid (LP Car): The acetone fraction dissolved in HCl was used and the absorbance was measured at 470 nm. The content of less polar carotenoid was calculated using Lichtenthaler equation. LP Car = (1000 A470 – 4.28 A665.4 – 4.78 653.4)/ 164 (g/ml) Results and DiscussionThe chlorophyll and its derivatives calculated for the five tree species collected from residential area and sugar mill were tabulated in table 1 and 2. All the five tree species showed reduction in chlorophyll a, b and carotenoids at the polluted site. Chlorophyll a, b and carotenoid of Ficus benghalensis was declined by 22.84%, 57.12% and 55.03% respectively near sugar mill. The percentage reduction of other species was also very high. The total chlorophyll and carotenoid of Delonix regia was decreased by 62.74% and 70.57% respectively. Higher percentage reductions were seen in Azadirachta indica (81.07% and 85.92%) and Pongamia pinnata (70.13% and 49.07%) for total chlorophyll and carotenoid. Ficus religiosa showed 45.73% decline in carotenoid pigment compared to the unpolluted site. The decomposed products of chlorophyll (protoporphyrin and magnesium-protoporphyrin) were formed more than hundred percent in Ficus benghalensis and Azadirachta indica near sugar mill compared to residential area. In Delonix regia, the percentage decompositions were 35.56% and 76.47% respectively. Pongamia pinnata showed 54.38% formation of International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202Vol. 1(4), 28-32, August (2012) I. Res. J. Biological Sci. International Science Congress Association 30 protoporphyrin and greater than hundred percent formation of magnesium-protoporphyrin. Another decomposition product protochlorophyllide (PChlide) was also formed in higher percentage for Ficus benghalensis, Delonix regia and Ficus religiosa where as less decomposed product than the residential area was reported in Azadirachta indica and Pongamia pinnata. The formation of chlide a and b were very low in both the polluted and unpolluted sites. The classical investigations of Willstatter and Stoll25 showed that when chlorophyll is treated with dilute acids there is formed a derivative, pheophytin, by quantitative replacement of the magnesium atom of the chlorophyll nucleus with hydrogen. Molisch26 found pheophytin to be present in gas damaged leaves27. Subsequently, Roben and Dorries28 in 1932 described experiments on the effect of sulfurous acid on Elodea canadensis in which the formation of pheophytin from chlorophyll was noted in shorter exposure periods, followed by bleaching of the green pigment and subsequent disappearance of the pheophytin as well, with time or at higher acid concentrations. It was also noted that sulfuric acid was far less damaging to the plant than sulfurous acid at equivalent hydrogen ion concentration. The pheophytin a and b were formed greater than hundred percent in Delonix regiaAzadirachta indica and Pongamia pinnata. For Ficus benghalensis, pheophytin a was found to be 27.17% and pheophytin b was more than hundred percent. In Ficus religiosa, pheophytin a was formed by 41.09% where as pheophytin b was produced less than the residential area. Less polar carotenoids was reported to be formed more than hundred percent in Ficus benghalensis and Delonix regia where as it was only 41.22% in Ficus religiosa. These results indicate that the decomposition of chlorophyll was higher near sugar mill because of the emission of gases26. The minimum decomposition of chlorophyll at the residential area was due to the natural autumnal yellowing or senescence of plants by organic acids released in the leaves in contact with the chlorophyll, as a result of the disintegration of plasma proteins29. Table 1 The chlorophyll and its derivatives for the five tree species at residential area Pigments Tree species Ficus benghalensis Delonix regiaFicus religiosaAzadirachta indica Pongamia pinnata Chl a 7.2624 13.4413 3.4549 21.2096 16.3653 Chl b 7.8518 8.2105 2.7814 8.0479 10.1246 Chl a+ b 15.1141 21.6518 6.2363 29.2575 26.4898 Car 4.8005 10.4651 4.5257 6.0144 4.7742 PPIX 16.8961 22.1329 18.8731 7.7797 29.4871 MGPP 7.3455 7.8210 5.9539 3.7771 9.6977 Pchlide 1.4307 0.0959 0.0707 0.2956 0.5897 Chlide a 0.0072 0.0062 0.0033 0.0045 0.0056 Chlide b 0.0069 0.0064 0.0044 0.0045 0.0064 Phe a 9.5859 8.0775 4.0579 6.1425 7.0022 Phe b 4.3818 4.5270 4.0257 2.3414 5.0729 LP Car 4.0427 4.4298 3.9569 1.1300 4.8460 Table 2 The chlorophyll and its derivatives for the five tree species near sugar mill Pigments Tree species Ficus benghalensis Delonix regiaFicus religiosaAzadirachta indica Pongamia pinnata Chl a 5.6035 4.7087 2.4545 3.8346 4.0656 Chl b 3.3668 3.3590 2.8738 1.7034 3.8482 Chl a+ b 8.9703 8.0677 5.3283 5.5380 7.9138 Car 2.1589 3.0799 2.4559 0.9367 2.4316 PPIX 64.0508 30.0043 12.0914 22.3197 45.5225 MGPP 19.0808 13.8019 4.8366 15.9559 19.5078 Pchlide 3.4422 3.8909 1.8781 9.4298 3.2889 Chlide a 0.0099 0.0167 0.0044 0.0261 0.0196 Chlide b 0.0125 0.0169 0.0049 0.0217 0.0207 Phe a 12.1901 22.4446 5.7253 36.0295 26.7548 Phe b 10.9589 11.8519 3.8524 11.4006 15.1852 LP Car 9.4571 17.7582 5.5878 11.0845 11.0446 International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202Vol. 1(4), 28-32, August (2012) I. Res. J. Biological Sci. International Science Congress Association 31 Chlorophyll a (Chl a) 18 Chlorophyll b (Chl a) Chlorophyllide a 19 Pheophytin a Chlorophyllide a Pheophytin a Pheophorbide a Pheophorbide b Figure-1 Degradation pathway of chlorophyll ConclusionAir pollution damage to vegetation produces a variety of gross symptoms on leaves which may have similar features as to color changes, location of lesions, and boundary characteristics30, 31. The order of susceptibility of various species to air pollution damage is a useful guide as to the probable causal agent. Chemical analysis of the leaf tissue provides additional help in diagnosis32, 33. The evidence in the present study on chlorophyll and pheophytin conversion in leaves of five tree species indicates that acid gases, such as sulfur dioxide in sufficiently high concentration, convert the chlorophyll pigments into the corresponding pheophytins. This process does not take place to an appreciable extent, however, unless the threshold value for sulfite in the living cells is exceeded. The effect may be produced by either relatively short exposures to high concentrations of gas or more prolonged exposures to lower concentrations. Transformation of an appreciable proportion of chlorophyll to pheophytin is accompanied by visible symptoms in the tissue, such as plasmolysis, shrinkage of cells, and fading of the normal green color. The destruction of the chlorophyll, pheophytin and other pigments in the chloroplasts results merely in the appearance of bleached or ivory colored areas. Sulfur dioxide absorption by leaves in subtoxic amounts produces no deleterious influence on photosynthesis and rate of growth34, 35. The gas is stored in the tissue largely as sulfate, and this may reach a value of 3 to 5 times the quantity normally present before it becomes toxic 36. Among the five tree species growing near sugar mill, Ficus religiosa showed minimum decomposition of chlorophyll and it was suggested to grow these species to reduce the load of pollution near sugar mills. References1.Sarala Thambavani D. and Sabitha M.A., Multivariate statistical analysis between COD and BOD of sugar mill effluent, Scholarly Journal of Mathematics and Computer Science,1(1), 6-12 (2012) 2.Sarala Thambavani D. and Sabitha M.A., Water quality and environmental assessment of sugar mill effluent, Journal of Research in Biology, , 125-135 (2012) 3.Bhosale P.R., Chonde S.G., Nakade D.B. and Raut P.D., Studies on Physico-Chemical Characteristics of Waxed and Dewaxed Pressmud and its effect on Water Holding Capacity of Soil, ISCA Journal of Biological Sciences, 1(1), 35-41 (2012) 4.Khwaja Mahmood A. and Quraishi Masood H., Self-Monitoring and Reporting for Industry Islamabad: Sustainable Development Policy Institute (SDPI) (2003)5.Muthusamy P., Murugan S. and Manothi Smitha, Removal of Nickel ion from Industrial Waste Water using Maize Cob, ISCA Journal of Biological Sciences, 1(2), 7-11 (2012) 6.Dohmen G.P., Koppers A. and Langebartels C., Biochemical response of Norway Spruce (Piceaabies (L.) Karst.) towards 14-month exposure to ozone and acid mist: effects on amino acid, glutathione and polyamine titers, Environmental Pollution, 64, 375-383 (1990) 7.Escobedo F.J., Wagner J.E., Nowak D.J., Analyzing the cost effectiveness of Santiago, Chile’s policy of using urban forests to improve air quality, Journal of Environmental Management, 86, 148-157 (2008)8.Hove L.W.A., Bossen M.E., Bok F.A.M., The uptake of O by poplar leaves: The impact of a long-term exposure to low O3- International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202Vol. 1(4), 28-32, August (2012) I. Res. J. Biological Sci. International Science Congress Association 32 concentrations, Atmospheric Environment, 33, 907-917 (1999) 9.Sarala Thambavani D. and Sabitha M.A., Variation in air pollution tolerance index and anticipated performance index of plants near a sugar factory: implications for landscape-plant species selection for industrial areas, Journal of Research in Biology, 1(6), (2011) 10.Joshi P.C. and Chauhan A., Performance of locally grown rice plants (Oryza sativa L.) exposed to air pollutants in rapidly growing industrial area of district Haridwar, Uttarakhand, India, Life Science Journal, 5(3), 57–61 (2008) 11.Hoque M.A., Banu M.N.A. and Okuma E., Exogenous proline and glycinebetaine increase NaCl-induced ascorbate glutathione cycle enzymes activities, and praline improves salt tolerance more than glycinebetaine in tobacco bright yellow -2 suspension – cultured cells, J. plant physiol., 164,1457–1468 (2007)12.Flowers M.D., Fiscus E.L. and Burkey K.O., Photosynthesis, chlorophyll fluorescence and yield of snap bean (Phaseolus vulgaris L) genotype differing in sensitivity to Ozone, Environ. Exp. Bot., 61, 190-198 (2007)13.Sarala Thambavani D. and Sabitha M.A., Tolerance of plants to air pollution near leather tanneries, Journal of Ecotoxicology and Environmental Monitoring, 1(6), 609-612 (2009a) 14.Klump G., Furlan C.M. and Domingos M., Response of stress indicators and growth parameters of tibouchina pulchra logn, Exposed to air and soil pollution near the industrial complex of cubatao, Brazil. Sci. Total Environ., 246, 79-91 (2000)15.Sarala Thambavani D., Sabitha M.A. and Selvasundari R., Air pollution Tolerance index of tree species growing in traffic area of Madurai, Tamil Nadu, The Asian Journal of Experimental Chemistry, 4(1&2), 126-132 (2009b) 16.Rao D.N., Plant leaf as pollution monitoring Device, Fertilizer News, 25-28 (1979)17.Reinbothe S. and Reinbothe C., Regulation of chlorophyll biosynthesis in angiosperm, Plant Physiol., 111, 1-7 (1996)18.Mcfeeters R.F., Chichester C.O. and Whitaker J.R., Purification and properties of chlorophyllase from Ailanthus altissima (Tree-of-heaven), Plant Physiol., 47, 609-618 (1971) 19.Lichtenthaler H.K., Chlorophylls and carotenoids: pigments of photosynthetic biomembrane, Methods Enzymol., 148, 350-382 (1987) 20.Porra R., Thompson W.A. and Kriedelman P.E., Determination of accurate extraction and simultaneously equation for assessing chlorophyll a and b extracted with different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy, Biochem. Biophys. Acta,975, 384-394 (1989)21.Holm G., Chlorophyll mutations in barley, Acta. Agric. Scand., , 457-461 (1954) 22.Khan V.M., Avivi-Bieise N. and Von Wettestein D., Genetic regulation of chlorophyll synthesis analyzed with double mutants in barley, In: Bhuchler T., (ed.), Genetics and Biogenesis of chloroplasts and mitochondria, Elseiver/North-Holland Biomedical Press Amsterdam, 119-131 (1976) 23.McFeeters R.F., Chichester C.O. and Whitaker J.R., Purification and properties of chlorophyllase from Ailanthus altissima (Tree-of-Heaven), Plant Physiol.,47, 609-618 (1971) 24.Lichtenthaler H.K., Chlorophylls and Carotenoids: pigments of photosynthetic biomembrane, Methods Enzymol., 148, 350-382 (1987) 25.Willstatter R. and Stoll A., Untersuchungen uber Chlorophyll, Julius Springer, Berlin (1913) 26.Molisch H., Mikrochemie der Pflanze, Auft., Gustav Fischer, Jena (1923)27.Mishra P.C., Dash A.K. and Pradhan Khageswar, Metals in Environmental segments at Hirakud of Odisha, India, ISCA Journal of Biological Sciences,1(1), 7-23 (2012)28.Roben M. and Dorries W., Berichte der Deutschen Botanischen Gessellschaft, 50, 53 (1932); Dorries W., Ibid., 50, 47 (1932)29.Wieler A., Jahrbuch fur wiss. Botani, 84, 764 (1937) 30.Srivastava K.P. and Singh Vikash Kumar, Impact of Air-Pollution on pH of soil of Saran, Bihar, India, Research Journal of Recent Sciences, 1(4), 9-13 (2012)31.Rathore Kanishka Raj, Dhawankar Aditi and Gungun, Environmental Impact Assessment (EIA) For Bus Based Rapid Transit System (BRTS) Bhopal, MP, India, Res. J. Recent Sci., 1, 166-171 (2012) 32.Chouhan Aarti, Iqbal Sanjeeda, Maheshwari R.S. and Bafna A., Study of air pollution tolerance Index of plants growing in Pithampur Industrial area sector 1, 2 and 3, Res.J.Recent Sci., , 172-177 (2012)33.Nwajei G.E., Okwagi P., Nwajei R.I. and Obi-Iyeke G.E., Analytical Assessment of Trace Elements in Soils, Tomato Leaves and Fruits in the Vicinity of Paint Industry, Nigeria, Research Journal of Recent Sciences, 1(4), 22-26 (2012) 34.Namdhari B.S., Rohilla S.K., Salar R.K., Gahlawat S.K., Bansal P. and Saran A.K., Decolorization of Reactive Blue MR, using Aspergillus species Isolated from Textile Waste Water, ISCA Journal of Biological Sciences, 1(1), 24-29 (2012)35.Murhekar Gopalkrushna Haribhau, Trace Metals Contamination of Surface Water Samples in and Around Akot City in Maharashtra, India, Research Journal of Recent Sciences, 1(7), 5-9 (2012). 36.Thomas M.D., Hendricks R.H. and Hill G.R., Proc. First Hational Air Pollution Symposium, 142, Stanford Res. Inst. (1949)