Studies on the degradation of coir pith using fresh water cyanobacterium Oscillatoria annae

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1 INT J CURR SCI, 7- RESEARCH ARTICLE ISSN -77 Studies on the degradation of coir pith using fresh water cyanobacterium Oscillatoria annae BDU and its lignolytic enzyme activity in response to coir pith degradation Abstract Anandhraj B, Krishna Moorthy S and Malliga P * Department of Marine Biotechnology, National Facility for Marine Cyanobacteria, Bharathidasan University Tiruchirapalli-, Tamil Nadu, India *Corresponding author: malli@yahoo.com; skrishmicro@gmail.com; Phone: +9-9 Lignin is an important constituent of plant materials and the second most abundant renewable polymeric component of biomass. Disposal of the recalcitrant is very difficult because of its lignocellulosic nature and thereby it s slow degradation in the natural environment. Cyanobacteria are free living photoautotrophic microorganisms that are widely distributed in the natural environment. Some cyanobacteria are able to fix atmospheric nitrogen and are therefore inexpensive to maintain. With such potentials they have been exploited in the field of lignocellulosic waste degradation, pesticide degradation, liquid waste treatment and degradation of aromatic compounds. Hence, the study mainly focused on the removal of coir pith waste through usual manner using fresh water cyanobacterium Oscillatoria annae BDU. The degradation of coir pith had been confirmed by the presence of lignolytic enzyme polyphenol oxidase activity exposed in the sample. The coir pith degradation was also confirmed by quantification of the following parameters such as reducing sugar, phenols, protein, lipids, nucleic acids, nitrate and ammonia. Keywords: coir pith, Oscillatoria annae, polyphenol oxidase Received: 9 th December; Revised: th January; Accepted: th January; IJCS New Liberty Group Introduction Cyanobacteria are free living photoautotrophic microorganism widely spread in soil, fresh water and marine habitats. They are gram negative, filamentous or unicellular grow as mats on the surface of bare soil as primary colonizers (Roger and Reynaud, 9). The combination of nitrogen fixing and non-nitrogen fixing algae give more effect in terms of growth and yield of paddy. The non-nitrogen fixing algae have also independently played an important role in the growth and yield of paddy. The combined effect of fixing and nonnitrogen fixing cyanobacteria have overall beneficial effects on soil enrichment, growth and yield of paddy (Selvarani, 9). Cyanobacteria are capable of abating various kinds of pollutants and have been used in the production of energy, fertilizer, human food, animal feed, polysaccharides, biochemicals and pharmaceuticals (Hall et al., 99). Coconut (Coccus nucifera Linn.) is one of the most useful and extensively cultivated palm in tropical countries. The waste products of coir yarn industry are coir dust and coir pith or coco peat which constitute about 7% of the husk. In spite of their limited use as soil conditioners, the quantity of coir dust produced is so enormous making its disposal difficult because of its lignocellulosic nature and slow degradation in the natural environment (Malliga et al., 99). Biodegradation is increasingly being considered as a less expensive alternative to physical and chemical means of decomposing organic pollutants. Bioremediation is valuable when cost control and system flexibility are needed (Alexander, 99). The enzymatic machinery for degrading cellulosic, hemicellulose and lignin is possessed only by microorganism (Rathor et al., ). Biological treatments of organic contamination are based on the degradative abilities of the microorganisms (Moreno and Becerra, ). Hence, the study focused on the removal coir pith using fresh water cyanobacterium Oscillatoria annae BDU and determination of the lignolytic activity of the cyanobacterium. Materials and methods Organism and culture Conditions A fresh water cyanobacterium belonging to Oscillatoria annae was obtained from the germplasm of

2 Anandhraj etal., National Facility for Marine Cyanobacteria, Bharathidasan University, Tiruchirappalli, Tamilnadu, India. The culture was maintained in BG medium (Rippka et al., 979), at lux. at ± C with / hrs light /dark cycle. Lignocellulosic Material Coir pith was collected from coir industry, near Srirangam, Tiruchirappalli, Tamil Nadu, India. Media and growth condition Cyanobacterial culture grown in BG medium were harvested, centrifuged and pooled in a different experimental media BG, NPK, Complex, Urea (Table ) and maintained in two different conditions one under white fluorescent light of, lux at ºC (laboratory condition) and another under that of, lux at 7 ºC (field condition). The cyanobacterial culture in BG medium acted as control. Table. Composition of Experimental medium Name of the Amount (g/) chemicals NPK medium NaNO. K HPO. H O. Complex medium Complex. Urea medium Urea. Biodegradation experiment with Oscillatoria annae: Inoculation and incubation In a series of ml conical flask, ml of BG medium was poured and g of cyanobacterial culture was inoculated, which was considered as positive control and to appropriate flask g of lignocellulosic material coir pith was added which is negative control, for test the medium was inoculated with the culture and coir pith in ratio:. The whole setup was done in triplicates and inoculation procedure was for the other three media (NPK, Complex and Urea). One set of the experimental setup was incubated under white fluorescent light of, lux at ºC (laboratory condition) and another set was maintained under, lux at 7 ºC (field condition). Qualitative and quantitative analysis On 7 th day, the cultures were centrifuged to separate pellet and supernatant and estimated. The spectral analysis, reducing sugar and phenol were analyzed in the supernatant. Pellets subjected to estimation of chlorophyll a, lignin, total lipids, quantification of DNA, RNA and enzyme study by Native Page. Results The first objective of the study was to select a growth media for the selected strain with minimal nutrients for degrading coir pith at large scale. Oscillatoria annae was capable of growing photoautotrophically in low nutrient and required only nitrogen source. Since it synthesizes its cellular material from carbon dioxide during photosynthesis, carbon source was not required in media. In this study, O. annae grown with coir pith in four different experimental media (BG, NPK, Complex and Urea), and BG medium acted as control. The cyanobacterium, O. annae grew well in all the media both under controlled and natural conditions and better growth of O. annae was obtained in Urea medium than other media such as BG, NPK and Complex. During the degradation of coir pith by Oscillatoria annae an appreciable amount of phenolic compounds were released into the surrounding medium and it was determined by spectral analysis at 7- nm wavelengths. Thus, spectrophotometric detection of peaks indicated the degradation of lignin and release of some interesting compounds into the medium (Figs. and ). The concentration of sugar considerably higher in coir pith degraded culture filtrate when compared with control cyanobacterium and coir pith (Figs.a and b) both in laboratory and field conditions. The figs. a and b showed that the phenol content also higher in coir pith treated cyanobacterial culture filtrate when compared to control in both in laboratory and field conditions. Similarly, O. annae grown with coir pith in urea medium exhibited more amount of phenol content compared to other media like BG, NPK and Complex. The Chlorophyll a content in control found to be lower when compared with test in both laboratory and field conditions in all the three media. The results also showed significant increase in chlorophyll a content of coir pith treated O. annae grown in BG, NPK, complex and urea media (Figs.a and b). While studying the

3 Anandhraj etal., biodegradation of coir pith by O. annae, it was observed that the reduction of lignin content in control coir pith was negligible, whereas, there was significant reduction of lignin content in test samples in all the three media used (Figs. a and b) both in laboratory and field conditions, however, urea medium showed maximum degradation of coir pith by O. annae when compared to all other media. Fig.. Estimation of sugar on 7 th day in (a) lab and (b) field condition compared to control cyanobacterium and coir pith alone (Figs. a and b). Polyphenol oxidase (PPO) activity of coir pith treated cyanobacterium, O. annae grown in different media showed induction of PPO activity when compared to control cyanobacterium alone. However, the induction in test samples grown in BG, NPK, complex and urea were similar (Fig ). Fig.. Estimation of phenol on 7 th day in (a) lab and (b) field condition a. Estimation of sugar on 7th day in lab condition a. Estimation of phenol on 7th day in lab condition BG NPK Complex Urea BG NPK Complex Urea b. Estimation of phenol on 7th day in field condition b.estimation of sugar on 7th day in field condition BG NPK Complex Urea BG NPK Complex Urea CB O. annae, Cp Coir pith, CB+Cp O. annae with coir pith Lipid analysis revealed that the lipid content was quantitatively higher in coir pith treated cyanobacterial sample grown in different experimental media like BG, NPK, Complex and Urea than in control cyanobacteria (Figs.7a and b) both in lab and field conditions. The figs. a, b and 9 a, b showed that coir pith treated cyanobacterial sample grown in urea medium contained higher amount of DNA and RNA when compared to other media. The nitrate and ammonia contents present in the supernatant were analyzed and from the result obtained, it is revealed that the amount of nitrate and ammonia were higher in coir pith treated cyanobacterial sample when compared to only cyanobacteria (Figs.a and b, a and b). The concentration of protein content in coir pith treated with cyanobacterial sample grown in all four media under laboratory and field conditions were higher when Discussion Cocos nucifera Linn. is the widely cultivated palm in the tropics. At present India is the principal producer and exporters of coir products. The by-products of coir industry are coir pith and coir dust which are the major waste as it gets accumulated enormously making its disposal difficult due to its high lignin content and slow degradation in the natural environment. In addition the environmental pollution due to the release of natural phenolic compounds from agro-industrial operations have become widespread in the world and utilization of these wastes is essential in the foreseeable future. Hence, this present investigation carried out to degrade the coir pith using a fresh water cyanobacterium, O. annae. The BG, NPK, complex and urea. The cyanobacterium O. annae grew well in all the media both under controlled and natural conditions and better growth of O. annae was obtained in Urea medium than other media

4 Anandhraj etal., like BG, NPK and Complex. Silva et al. (7) reported the optimization of mineral nutrition in the culture medium showed increased final biomass level and higher flocculation rates of Tolypothrix tenuis. This could be the reason for increase in biomass of cyanobacteria when grown in urea media. The degradation of coir pith by cyanobacterium was determined by spectral analysis. The spectral results showed that the selected strain Oscillatoria annae has the ability to degrade the lignocellulosic waste coir pith within a short period of incubation (7 days). This present finding correlates with that of Malliga et al. (99); Vendramani and Trugo, () they studied the phenolic compounds in acerola fruit (Malpighia punicifolia L.) and reported that the absorbance in UV region denoted that the compound released is of aromatic nature. The the supporting evidence for the lignolytic activity of O. annae. The presence of reducing sugar might be due to cleavage of complex polymers to simple sugars by the cyanobacterial enzyme. The result coincides with the previous studies made by Shasirekha et al. () reported the release of reducing sugars and amino acids into the media during the growth of Pleurotus florida on rice straw. Degradation of coir pith resulted in release of some organic compounds such as phenol which resulted in change in color of media from colorless to brown. The source of the phenol released into the supernatant is the lignin moiety which is a polymer of phenolic compounds. The result coincides with that result of the spectral analysis, which confirmed that the absorbance in the UV region was due to the release of phenolic compounds. presence of reducing sugars in the supernatant acted as Fig.. Estimation of phenol on 7 th day in (a) lab and (b) Fig. b. Estimation of lignin on 7 th day in field condition field condition b. Estimation of lignin on 7th day in field condition Chl a content (mg/ml) a. Estimation of chlorophyll a on 7th day in lab conditon BG NPK Complex Urea % of lignin 7 Cp CB+Cp BG N P K Complex Urea Chl a content (mg/ml) b. Estimation of chlorophyll a on 7th day in field condition BG NPK Complex Urea CB O. annae, CB+Cp O. annae with coir pith Fig. a. Estimation of lignin on 7 th day in lab condition CB O. annae, Cp Coir pith, CB+Cp O. annae with coir pith Fig. 7a. Estimation of phenol on 7 th day in (a) lab and (b) field condition a. Estimation of lipids on 7th day in lab condition a. Estimation of lignin on 7th day in lab condition BG NPK Complex Urea 7. b. Estimation of lipids on 7th day in field condition % of lignin Cp CB+Cp..... BG N P K Complex Urea BG- NPK Complex Urea

5 Anandhraj etal., Fig.. Spectral analysis of different media on 7 th day grown with O. annae with coir pith under laboratory condition CB+CP Cyanobacteria + Coir Pith, CP Coir Pith, CB Cyanobacteria. Lab and BG Lab and complex... Lab and NPK Lab and Urea Fig..Spectral analysis of different media on 7 th day grown with O. annae with coir pith under field condition CB+CP Cyanobacteria + Coir Pith, CP Coir Pith, CB Cyanobacteria. Filed and BG Field and complex. Field and NPK Field and Urea

6 Anandhraj etal., Fig.. Estimation of DNA on 7 th day in (a) lab and (b) field condition (CB O. annae, CB+Cp O. annae with coir pith) a. Estimation of DNA on 7th day in lab conditon 7 b. Estimation of DNA on 7th day in field Condition BG NPK Complex Urea BG NPK Complex Urea Fig. 9. Estimation of RNA on 7 th day in (a) lab and (b) field condition (CB O. annae, CB+Cp O. annae with coir pith) a. Estimation of RNA on 7th day in lab condition b. Estimation of RNA on 7th day in field condition BG NPK Complex Urea BG NPK Complex Urea Fig.. Estimation of nitrate on 7 th day in (a) lab and (b) field condition (CB O. annae, CB+Cp O. annae with coir pith) a. Estimation of nitrate on 7th day in lab condition BG NPK SamplesComplex Urea b. Estimation of nitrate on 7th day in field condition BG NPK Complex Urea Fig.. Estimation of ammonia on 7 th day in (a) lab and (b) field condition 9 7 a. Estimation of ammonia on 7th day in lab condition BG NPK Complex Urea 9 7 b. Estimation of ammonia on 7th day in field condition BG NPK Complex Urea

7 Anandhraj etal., From the above result, it was clear that O. annae degraded the lignocellulosic material coir pith containing lignin and holocellulose which was so far proved by other microorganisms such as bacteria (Perestelo et al., 99), actinomycetes (Ferraz and Duran, 99) and fungi (Bhat and Narayan, ). Fig.. Estimation of protein on 7 th day in (a) lab and (b) field condition (CB O. annae, Cp Coir pith, CB+Cp O. annae with coir pith) a. Estimation of protein on 7th day in lab condition BG NPK Complex Urea b. Estimation of protein on 7th day in field condition BG NPK Complex Urea Fig. Induction of Polyphenol oxidase on 7 th day incubation of Oscillatoria annae with coir pith by native gel electrophoresis (Cyanobacteria only: C BG, C NPK, C Complex, C Urea; Cyanobacteria + Coir Pith: T BG, T NPK, T Complex, T Urea) The chlorophyll content increased when cyanobacterium inoculated with coir pith. Supporting evidence proved that the growth of a cyanobacterium namely Anabaena azollae ML with coir pith showed an increase in growth rate in terms of chlorophyll a (Malliga et al., 99). Thus it can be concluded that the increased chlorophyll content in test sample indicated that the presence of coir pith was not inhibiting the growth of O. annae and interestingly O. annae degraded the coir pith and utilized coir pith as a source of nutrient for its growth and other metabolic activities. The presence of lignin level in all the four experimental media was decreased. The decreased amount of lignin is the indication of the lignin degradation by cyanobacterium. But, urea medium showed maximum degradation of coir pith by O. annae when compared to other media. It could be due to the fact that urea acted as nitrogen source to O. annae in the simplest form, comparing the other three media, which would in turn help in higher metabolic rate. These results correlated with an investigation made by Wu et al. () to explore the lignin degrading capacity of five white rot fungi Phanerochaete chrysosporium, Pleurotus ostreatus, Lentinus edodes, Trametes versicolor and S reported that they were individually used to treat black liquor from pulp and paper mill over 7% of lignin and % of COD removed from the waste water in days. The lipid content in the experimental media which inoculated by cyanobacterial culture with coir pith was higher when compared to control. The maximum content of lipid was observed in urea medium. This report fell in accordance with Hung et al. () who reported that the Isochrysis galbana when grown in urea culture gave the highest biomass yield and lipid content of.±.g/l and.±.% respectively after hrs of cultivation. Malliga (99) reported that the nucleic acids content, both DNA and RNA, increased in Azolla pinnata on exposure to varying nitrogen content in different media like nitrogen free media, sodium nitrtate, ammonium chloride and ammonium nitrate. Azolla pinnata showed maximum nucleic acid content in ammonium nitrate media. The response could be due to the fact that ammonium nitrate media provided the most easily accessible nitrogen. Similarly, in this study urea medium consisted of nitrogen in the simplest form and correspondingly O. annae grown in urea medium showed the maximum nucleic acids content. The nitrate and ammonia contents were higher in the supernatant of urea

8 Anandhraj etal., medium which inoculated by cyanobacteria with coir pith. These results supported by Stal and Krumbein (9) who reported the fixation of nitrogen under aerobic conditions by the non-heterocystous cyanobacterium Oscillatoria sp. strain. According to them Oscillatoria sp. strain while grown under alternating light-dark cycles, fixes N preferable in the dark period. Providing light-dark condition in lab and natural light-dark condition in field during the degradation of coir pith by O. annae could have resulted in the nitrogen fixation resulting in accumulation of nitrate and ammonia in media. The increase in protein content of test samples could be due to the induction of lignolytic enzymes, required for the degradation of lignin present in coir pith. Shashirekha et al., (997) determined the growth of Phormidium valderianum during phenol degradation in terms of protein content and observed that test samples showed higher protein content on exposure to phenol. Their report fell in accordance with this study since lignin is phenolic polymer and exposure to coir pith induced increase in protein content. The induction of polyphenol oxidase was determined by the native gel electrophoresis. The induction of the enzyme occurred in all the experimental media which inoculated with coir pith. The brown band that appeared confirmed polyphenol oxidase activity which is constitutive enzyme (Bligny and Douce, 9). Induction of two isoforms of polyphenol oxidase on exposure to lignocellulose indicated that the polyphenol oxidase is a inducible enzyme. Prabha, () reported that the presence of polyphenol oxidase activity of O. laetevirens on P. juliflora. These results are also correlated with lignolytic enzyme profile of Oscillatoria annae was studied by Viswajith () for justifying the lignin degrading ability of O. annae. The results revealed the presence of manganese independent peroxidase, laccase, polyphenol oxidase and other cellulolytic enzymes like endogluconase and xylanase. Also H O production was markedly enhanced in O. annae exposed to lignocellulosic waste, which indicates the increase in ligninolytic activity characteristic of cultures grown under stress conditions. Conclusions The degradative ability of cyanobacterium (Oscillatoria annae) is due to the strong oxidative activity and the low substrate specificity of their ligninolytic enzymes which was confirmed by induction in polyphenol oxidase activity in cyanobacteria exposed to coir pith containing experimental media. References Alexander M (99). Bio-degradation kind bioremediation academic, San Diego, Calif., pp -7, -. Bhat AD, P Narayanan (). Chromatographic analysis of phenolics and study of Klason lignin biodegraded coir pith using Pleurotus sajor-caju. Dissertation, University of Kerala, India. Chia-Hung Su, Giridhar R, Chiao-wei chen, Wen-Teng Wu (). A novel approach for medium formulation for growth of a microalga using motile intensity. Bioresource technology. 9 (): -. Ferraz A, N Duran (99). Lignin degradation during softwood decaying by the ascomycete Chrysonilia sitophila. Biodegradation. (): -7. Hall DO, Markov SR Watanable Y, Rao KK. (99). The potential applications of cyanobacterial photosynthesis in clean technologies. Photosynthesis research. : 9 7. Malliga P (99). Microbial association in the leaf cavities of Azolla pinnata R.Br. Ph.D Dissertation submitted to Bharathidasan University, Trichirappalli, Tamil Nadu, India. Malliga P, Uma L, Subramnian G (99). Lignolytic activity of the cyanobacterium Anabaena azollae ML and the value of coir waste as a carrier for biofertilizer. Microbios. : 7-. Perestelo F, MA, Falcon A, Carnicero A, Rodriguez, DLG Feunte (99). Limited degradation of industrial, synthetic and natural lignin by Serratia marcescens. Biotechnol. Lett. : 99-. Prabha DS (). Studies on the degradation of Prosopis juliflora (Sw.) D.C. by marine cyanobacterium (Oscillatoria laetevirens) and

9 Anandhraj etal., its assessment of bioactive property on animal model. Ph.D. Dissertation submitted to Department of Marine biotechnology, NFMC, Bharathidasan University, Tiruchirappalli-, Tamil Nadu, India. Rathore V, L Kumar, H Srivastava (). C-[lignin]- lignocellulose biodegradation by bacteria isolated from polluted soil. Ind. J. Exp. Biol. 9(): -9. Rippka R, J Deruelles, JB Waterbury, M Herdman, RY Stainer (979). Genetic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. : -. Roger PA, Reynaud PA (9). Free living blue green algae in tropical soils. In: microbiology of tropical soils and plant productivity. (Ed) Y.R. Dommergues, HG Diem, Pp. 7-. Selvarani V (9). Studies on the influence on nitrogen fixing and non-nitrogen fixing blue-green algae on the soil, growth and field of paddy (Oryza sativa-tr ). Shashirekha MN, S Rajarathnam, Z Bano (). Effects of supplementing rice straw growth substrate with cotton seeds on the analytical characteristics of the mushroom Pleurotus florida. Food Chem. 9(): -9. Silva AMM, AR Silva, AM Pinheiro, SRVB Freitas, V DA, Silva CS, Souza JB, Hughes, SL, Costa (7). Alkaloids from Prosopis juliflora leaves induce glial activation, cytotoxicity and stimulate NO production. Toxicon. 9 (): -. Stal JL, Krumbein WE (9). Nitrogenase activity in the non-heterocystous cyanobacterium Oscillatoria sp. grown under alternating light dark cycles. Archives of Microbiol. (): 7-7. Vendramani ALA, LC Trugo (). Phenolic compounds in acerola fruit (Malpighia punicifolia L.). J. Braz. Chem. Soc. : - Wu J, YZ. Xiao, HQ Yu (). Degradation of lignin in pulp mill wastes waters by white rot fungi on biofilm. Biores. Technol. 9(): 7-. Viswajith V, P Malliga (). Ligninolytic enzyme profile of Oscillatoria annae in response of Lantana camara. Indian J. Bot. Res.:7-7.