- Email: [email protected]
Removal of nutrients and organic pollution load from pulp and paper mill effluent by microalgae in outdoor open pond
Accepted Manuscript Removal of nutrients and organic pollution load from pulp and paper mill effluent by microalgae in outdoor open pond M.T. Usha, T. Sarat Chandra, R. Sarada, V.S. Chauhan PII: DOI: Reference:
S0960-8524(16)30547-8 http://dx.doi.org/10.1016/j.biortech.2016.04.060 BITE 16419
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
19 March 2016 13 April 2016 15 April 2016
Please cite this article as: Usha, M.T., Sarat Chandra, T., Sarada, R., Chauhan, V.S., Removal of nutrients and organic pollution load from pulp and paper mill effluent by microalgae in outdoor open pond, Bioresource Technology (2016), doi: http://dx.doi.org/10.1016/j.biortech.2016.04.060
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Removal of nutrients and organic pollution load from pulp and paper mill effluent by microalgae in outdoor open pond M. T. Usha, T. Sarat Chandra, R. Sarada, V. S. Chauhan* Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Institute, Mysuru - 570020, Karnataka, India
*Corresponding Author Email: [email protected] Telephone - +91 821 2516501 Tele-fax - +91-821-2517233
1.0 Introduction The requirement of large amount of water and nutrient for microalgal cultivation is a major bottleneck in realisation of microalgae based biofuel production. However the ability of algae to grow in non-potable waters such as sewage water and secondary treated industrial waste waters could be exploited for long-term sustainability (Yang et al., 2011). Utilisation of wastewater for microalgal cultivation serves the dual purpose of supply of nutrients and minimisation of fresh water requirements along with the concomitant reduction of COD, BOD from the wastewater. Microalgae are versatile and can grow either by autotrophic or mixotrophic modes of nutrition. Concurrent assimilation of organic and inorganic carbon may result in the mixotrophic growth of algal cells (Li et al., 2011). Cultivation of microalgae by mixotrophic mode of nutrition overcomes the limitation of light requirement presented during the photoautotrophic mode of nutrition. Thus, algae can utilise high levels of organic carbon from wastewater for faster growth under the photo- heterotrophic or mixotrophic conditions in presence of light (Li et al., 2011). Microalgae have also been reported for their ability to remove metals from the wastewater. Several studies have reported the utilisation of wastewater for cultivation of microalgae (Komolafe et al., 2014; Ramanna et al., 2014). Most of the studies have reported the utilisation of sewage water (Ji et al, 2014; Cho et al., 2013) and dairy wastewater (Beevi and Sukumaran, 2014) for microalgal cultivation. Other effluents such as swine effluent (Wu et al., 2013; Travieso et al., 2006), anaerobic digestate (Uggetti et al., 2014,; Ji et al., 2014) and brewery effluent (Mata et al., 2014) have also been extensively studied for microalgal cultivation. Gentili et al. (2014) have discussed the possibility of mixing different types of wastewaters for production of algal biomass. However, the use of pulp and paper effluent for the cultivation of microalgae is least explored (Tarlan et al., 2002; Gentili, 2014). The pulp and paper industry is one of the largest producers of plant-based wastewater in the world. About 10- 50 m3 of water is required to produce a tonne of paper. In this scenario, a large amount of wastewater is generated which
has to be treated before its release. The conventional treatment of pulp and paper effluent requires the addition of nutrients as the effluent is limited in nitrogen and phosphorous (Slade et al., 2004). In the present study utilisation of primary treated pulp and paper effluent for the cultivation of a mixed culture of microalgae containing two Scenedesmus sps. was investigated. 2.0 Materials and Methods 2.1 Microalgal culture A mixed culture of microalgae containing two Scenedesmus sp. isolated by Vidyashankar et al. (2103) was used in this study. Two species were initially cultivated individually in Bold Basal Media (Vidyashankar et al., 2013) under controlled conditions at 25oC, 250 µ mol m2 s-1 light intensity under 16:8 light/ dark cycle. 2.2 Wastewater The primary treated wastewater was procured from M/s South Indian Paper Mill located in Mysore, Karnataka, India. The wastewater was carried to the laboratory and was stored in tightly capped glass carboys in cold storage (4oC) for further use. The physico-chemical parameters of wastewater were analysed as per the standard methods (APHA, 1998). For batch studies to assess the effect of wastewater on microalgal growth, the wastewater was diluted with distilled water to obtain the required concentration (5%, 10%, 20%, 40%, 60%, 80%, and 100%). 2.3 Experimental set up The microalgal cultivation studies were conducted in outdoor open circular ponds (1m x 1.5m) of 30L capacity for 28 days. The intermittent manual mixing was provided to avoid the sedimentation of microalgae. During the study, the ambient temperature and the light
intensity were observed to be 23±3 oC and 0.8- 1.8 Kilo lux. The light intensity was measured using digital lux metre (TES 1332, TES Electrical Electronic Corp, Taiwan). Samples were withdrawn at regular intervals and was analysed for NO3-N, PO4-P, iron, zinc, Chemical oxygen demand (COD), biological oxygen demand (BOD) and total organic carbon (TOC). COD, BOD, nitrate and phosphate analysis was carried out as per the standard methods (APHA, 1998). Iron and zinc content was analysed using Atomic Absorption Spectroscopy (AAS). TOC was analysed by TOC analyser. All the experiments were carried out in duplicates. Once the microalgal cultures reached the stationary phase, they were harvested by centrifugation and the obtained biomass were sundried. The lipids from dried biomass were extracted (Moisture content- 6% (w/w)) with hexane using Soxhlet apparatus (Sarat Chandra et al., 2016). Further, Fatty Acid Methyl Ester (FAME) analysis was conducted as per the protocol described in Sarat Chandra et al. (2016) 2.4 Determination of microalgal growth The microalgal growth was determined by measuring the optical density (OD) of culture at 560 nm using a UV/ Visible spectrophotometer (Model: UV/1800A, Shimadzu Corporation, Kyoto, Japan). A relationship between optical density and biomass concentration (g L-1) was established using a calibration curve and it was found that OD560 value of 1 was equivalent to 0.7 g L-1 of biomass. 3.0 Results and Discussion 3.1 Wastewater characterisation The wastewater was characterised for its physico-chemical properties. The pH, conductivity and alkalinity of wastewater were 5.41±0.56, 3932±10 µs cm-1 and 228±4.29 mg L-1. The Total Solids (TS) content of the wastewater was 6446±23.24 mg L-1 with 3580±10.23 mg L-
1
of Total Dissolved Solids (TDS), and 2866±16.56 mg L-1 of Total Suspended Solids (TSS).
The COD, BOD and TOC of the wastewater were 3000.15±28.15 mg L-1, 2944±12.34 mg L-1 and 198±8 mg L-1 respectively. The nitrogen, phosphorous and sulphur are the major nutrients required for the growth of microalgae. A C: N: P ratio of 46.1: 7.7: 1 has been reported as optimal for the growth of microalgae (Hillebrand and Sommer, 1999). The N: P ratio of the paper and pulp wastewater in the present study was 1:3 with the total nitrate being 9.932±1.87 mg L-1 and phosphate being 30.25±3.28 mg L-1. The sulphates (86.48±2.56 mg L-1) and other media constituents such as iron (9.729±0.47 mg L-1) and zinc (7.23±0.38 mg L1
) were found to be sufficient to support the growth of algae. The wastewater also showed a
chloride content of 204±5.68 mg L-1. 3.2 Indoor laboratory studies The indoor microalgal growth studies were conducted in laboratory using 250ml Erlenmeyer flasks. The microalgae were cultivated with various concentrations of wastewater (5%, 10%, 20%, 40%, 60%, 80% and 100%). The microalgal growth was recorded at regular intervals in terms of OD560. The growth profile of microalgal culture at various concentrations of wastewater is shown in Figure 1a. It is evident from Figure 1a that with the increase in the concentration of wastewater (upto 60%), the microalgal growth also increased and was observed to be better than control cultures grown in Bold Basal Medium. However, the microalgal growth was marginally inhibited at wastewater concentrations of > 60% . Therefore, the 60% wastewater (60:40 dilution of wastewater with water) was considered as optimum for microalgal cultivation. 3.3 Wastewater treatment by mixed microalgal culture in outdoor open pond Based on the indoor laboratory studies, 60% concentration of wastewater was chosen as optimum and further studies were conducted in outdoor open ponds. The outdoor open pond cultivation study was carried out for 28 days in a batch mode. The pH of the culture was
monitored on a regular basis throughout the study period and was in the range of 8-10. The biomass was partly harvested when the cultures reached the stationary phase on 16th day and wastewater was recycled. The biomass was harvested at the end of the study period on 28th day. The growth profile of the mixed microalgal culture in outdoor open pond is given in Figure. 1b. The drop in the OD on the 16th day was due to the harvesting of biomass. The study suggests that paper and pulp wastewater could support the long duration microalgal cultivation. 3.4 Nutrient removal Microalgae have the potential for the removal of nutrients from wastewater and cultivation of microalgae in wastewater is a cost effective approach for remediation of organic load and nutrients. The efficiency of the effluent treatment by microalgae is related to its ability to reduce the COD, BOD, nitrogen and phosphorous levels in the effluent. The profile of COD, TOC and BOD removal is shown in Figure 2a. Microalgal cultivation in the effluent resulted in 89% and 75% removal of COD and BOD respectively. Nitrogen and phosphorous are the major nutrients required for microalgal growth, as both nitrogen and phosphorous found in nucleic acids, proteins, carbohydrates and the intermediates of carbohydrate metabolism. The removal of nitrate and phosphate was analysed by periodic determination of NO3-N and PO4P levels in the wastewater used for the cultivation of microalgae in outdoor open pond. The profile of NO3-N and PO4-P removal from wastewater by microalgae is showed in the Figure 2b. On 28th day of microalgal cultivation, 65% and 71.29% removal of NO3-N and PO4-P, respectively, was achieved. Iron and zinc are the micronutrients required for the growth of the microalgae and at the end of 28 days of microalgal cultivation, 25% and 68% removal of iron and zinc respectively from wastewater was recorded (Figure. 2c). Hongyang et al. (2011) has reported 77.8%, 88.8% and 70.3% COD, nitrogen and phosphorus removal respectively from soybean processing wastewater via cultivation of microalga Chlorella pyrenoidosa. An 88% COD removal rate of piggery wastewater by Chlorella sps has been
reported by Travieso et al. (2006) for an initial COD of 250 mg L-1 at 190 hrs which is much higher than obtained in the present study (34% COD reduction for an initial COD of 1520 mg L-1 in 190 hrs). This may be attributed to the difference in microalgal species and the wastewater considered. 3.5 Biomass characterization Microalgal biomass was harvested from outdoor open pond at the end of the cultivation period of 28 days and analysed for its biochemical composition. The microalgal biomass was found to be rich in protein (16±2.45%), iron (2.48 mg g-1) and zinc (0.366 mg g-1). The carbohydrate and lipid content (% wt/wt) of the biomass were 18.90±3.2% and 15.8±4% respectively. The biomass was also analysed for its fatty acid composition (Figure 3). The palmitic acid (C16:0), alpha linolenic acid (C 18:3) and oleic acid (C 18:2) were the major fatty acids contributing upto 32%, 24% and 18.52 respectively in the biomass harvested on 16th day. The alpha linolenic acid, palmitic acid, oleic acid and linoleic acid were major fatty acids contributing upto 31%, 29%, 13% and 13% respectively in the biomass harvested on 28th day. Therefore, the study suggests that the microalgal biomass thus produced utilising the paper and pulp wastewater has the potential for food, feed applications. 4.0 Conclusion Microalgae for removal of nutrients and organic load from pulp and paper mill effluent was studied. A removal of upto 89% and 75% of COD and BOD respectively was achieved. NO3-N and PO4-P removal of upto 65% and 71.29% respectively was observed at the end of 28 days. The biomass obtained was found to be rich in protein and also showed significant presence of α-linolenic acid. Therefore, the present study showed that microalgae can be effectively used for removal of nutrients and organic matter from paper and pulp mill wastewater with concomitant production of microalgal biomass for value added application.
Acknowledgements Financial support (BT/PR1311/PBD/26/267/2011) from Department of Biotechnology (DBT), Government of India is gratefully acknowledged. TSC is thankful to Council of Scientific and Industrial Research (CSIR) for providing research fellowship. The authors thank Director, CSIR-CFTRI for encouragement. References 1) American Public Health Association (APHA), 1998. Standard methods for the examination of water and wastewater, 20th ed. APHA AWWA WPCF, Washington, DC. 2) Beevi, U. S., Sukumaran, R. K., 2014. Cultivation of microalgae in dairy effluent for oil production and removal of organic pollution load. Bioresource Technology. 165, 295301. 3) Cho, S., Lee, N., Park, S., Yu, J., Luong, T. T., Oh, Y., Lee, T., 2013. Microalgae cultivation for bioenergy production using wastewaters from a municipal WWTP as nutritional sources. Bioresource Technology. 131, 515- 520. 4) Gentili, F. G., 2014. Microalgal biomass and lipid production in mixed municipal, dairy, pulp and paper wastewater together with added flue gases. Bioresource Technology. 169, 27- 32. 5) Hillebrand, H., Sommer, U., 1999. The nutrient stoichiometry of benthic microalgal growth: redfield proportions are optimal. Limnology and Oceanography 44, 440- 446. 6) Hongyang, Su., Yalei, Z., Chunmin, Z., Xuefei, Z., Jinpeng, L., 2011. Cultivation of Chlorella pyrenoidosa in soybean processing wastewater. Bioresource Technology. 102(21), 9884-9890. 7) Ji, F., Liu, Y., Hao, R., Li, G., Zhou, Y., Dong, R., 2014. Biomass production and nutrients removal by a new microalgae strain Desmodesmus sp. in anaerobic digestion wastewater. Bioresource Technology. 161, 200- 207.
8) Ji, M.K., Kabra, A. N., Salama, E.S., Roh, H.S., Kim, J. R., Jeon, B., Lee, D. S., 2014. Effect of mine wastewater on nutrient removal and lipid production by a green microalga Micratinium reisseri from concentrated municipal wastewater. Bioresource Technology. 157, 84- 90. 9) Komolafe, O., Orta, S. B. V., Monje-Ramirez, I., Noguez, I. Y., Harvey, A. P., Ledesma, M. T. O., 2014. Biodiesel production from indigenous microalgae grown in wastewater. Bioresource Technology. 154, 297- 304.
10) Li, Y., Chen, Y.-F., Chen, P., Min, M., Zhou, W., Blanca, M., 2011. Characterization of a microalga Chlorella sp. well adapted to highly concentrated municipal wastewater for nutrient removal and biodiesel production. Bioresource Technology. 102, 5138–5144. 11) Mata, T. M., Mendes, A. M., Caetano, N. S., Martins, A. A., 2014. Sustainability and economic evaluation of microalgae grown in brewery wastewater. Bioresource Technology. 168, 151-158. 12) Ramanna, L., Guldhe, A., Rawat, I., Bux, F., 2014. The optimization of biomass and lipid yields of Chlorella sorokiniana when using wastewater supplemented with different nitrogen sources. Bioresource Technology. 168, 127- 135.. 13) Sarat Chandra, T., Deepak, R. S., Maneesh Kumar, M., Mukherji, S., Chauhan, V. S., Sarada, R., Mudliar, S. N., 2016. Evaluation of indigenous fresh water microalga Scenedesmus obtusus for feed and fuel applications: Effect of Carbon dioxide, light and nutrient sources on growth and biochemical characteristics. Bioresource Technology. 207, 430-439. 14) Slade, A.H., Ellis, R.J., vanden Heuvel, M., Stuthridge, T.R., 2004. Nutrient minimisation in the pulp and paper industry: an overview. Water Science and Technology. 50, 111– 122 15) Tarlan, E., Dilek, F.B., Yetis, U., 2002. Effectiveness of algae in the treatment of a woodbased pulp and paper industry wastewater. Bioresource Technology. 84, 1–5
16) Travieso, L., Benitez, F., Sanchez, E., Borja, R., Martin, A., Colmenarejo, M. F., 2006. Batch mixed culture of Chlorella vulgaris using settled and diluted piggery waste. Ecological Engineering. 28(2), 158-165. 17) Uggetti, E., Sialve, B., Latrille, E., Steyer, J., 2014. Anaerobic digestate as substrate for microalgae culture: The role of ammonium concentration on the microalgae productivity. Bioresource Technology. 152, 437-443.
18) Vidyashankar, S., Deviprasad, K., Chauhan, V. S., Ravishankar, G. A., Sarada, R., 2013. Selection and evaluation of CO2 tolerant indigenous microalga Scenedesmus dimorphus for unsaturated fatty acid rich lipid production under different culture conditions. Bioresource Technology. 144, 28–37. 19) Wu, P., Teng, J., Lin, Y., Hwang, S, J., 2013. Increasing algal biofuel production using Nannocholropsis oculata cultivated with anaerobically and aerobically treated swine wastewater. Bioresource Technology. 133, 102- 108. 20) Yang, J., Xu, M., Zhang, X., Hu, Q., Sommerfeld, M., Chen, Y., 2011. Life-cycle analysis on biodiesel production from microalgae: Water foot print and nutrient balance. Bioresource Technology. 102, 159-165.
Figure Captions Figure 1 (a) Growth profile of mixed microalgal culture at various concentrations of pulp and paper mill effluent in laboratory, and (b) Growth profile of mixed microalgal culture in outdoor open pond with 60% pulp and paper mill effluent. Figure 2 Profile of (a) COD, BOD and TOC removal (b) Nitrate and phosphate removal and (c) iron and zinc removal from pulp and paper mill effluent utilised for cultivation of microalgae in outdoor open pond. Figure 3 Fatty acid profile of mixed microalgal culture cultivated with pulp and paper mill effluent.
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Highlights
COD and BOD removal of upto 89% and 75% respectively was achieved. NO3-N and PO4-P removal of upto 65% and 71.29% respectively was observed. An enhanced growth of microalgae was observed with 60% effluent. The palmitic acid and α-linolenic acid were the major fatty acids of microalgae.
Keywords: pulp and paper mill effleunt, Scenedesmus, COD, Lipids, nutrient removal
Abstract: A mixed culture of microalgae, containing two Scenedesmus species, was analysed to determine its potential in coupling of pulp and paper mill effluent treatment and microalgal cultivation. Laboratory studies suggested that 60% concentration of wastewater resulted in better biomass growth than the control. A maximum of 89% and 75% removal of COD and BOD respectively was achieved with microalgal cultivation in outdoor open pond. By the end of the cultivation period, 65% removal of NO3-N and 71.29% removal of PO4-P was observed. The fatty acid composition of mixed microalgal culture cultivated with effluent showed the palmitic acid, oleic acid, linoleic acid and α-linolenic acid as major fatty acids. The results obtained suggest that pulp and paper mill effluent could be used effectively for cultivation of microalgae to minimise the freshwater and nutrient requirements.
S0960-8524(16)30547-8 http://dx.doi.org/10.1016/j.biortech.2016.04.060 BITE 16419
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
19 March 2016 13 April 2016 15 April 2016
Please cite this article as: Usha, M.T., Sarat Chandra, T., Sarada, R., Chauhan, V.S., Removal of nutrients and organic pollution load from pulp and paper mill effluent by microalgae in outdoor open pond, Bioresource Technology (2016), doi: http://dx.doi.org/10.1016/j.biortech.2016.04.060
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Removal of nutrients and organic pollution load from pulp and paper mill effluent by microalgae in outdoor open pond M. T. Usha, T. Sarat Chandra, R. Sarada, V. S. Chauhan* Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Institute, Mysuru - 570020, Karnataka, India
*Corresponding Author Email: [email protected] Telephone - +91 821 2516501 Tele-fax - +91-821-2517233
1.0 Introduction The requirement of large amount of water and nutrient for microalgal cultivation is a major bottleneck in realisation of microalgae based biofuel production. However the ability of algae to grow in non-potable waters such as sewage water and secondary treated industrial waste waters could be exploited for long-term sustainability (Yang et al., 2011). Utilisation of wastewater for microalgal cultivation serves the dual purpose of supply of nutrients and minimisation of fresh water requirements along with the concomitant reduction of COD, BOD from the wastewater. Microalgae are versatile and can grow either by autotrophic or mixotrophic modes of nutrition. Concurrent assimilation of organic and inorganic carbon may result in the mixotrophic growth of algal cells (Li et al., 2011). Cultivation of microalgae by mixotrophic mode of nutrition overcomes the limitation of light requirement presented during the photoautotrophic mode of nutrition. Thus, algae can utilise high levels of organic carbon from wastewater for faster growth under the photo- heterotrophic or mixotrophic conditions in presence of light (Li et al., 2011). Microalgae have also been reported for their ability to remove metals from the wastewater. Several studies have reported the utilisation of wastewater for cultivation of microalgae (Komolafe et al., 2014; Ramanna et al., 2014). Most of the studies have reported the utilisation of sewage water (Ji et al, 2014; Cho et al., 2013) and dairy wastewater (Beevi and Sukumaran, 2014) for microalgal cultivation. Other effluents such as swine effluent (Wu et al., 2013; Travieso et al., 2006), anaerobic digestate (Uggetti et al., 2014,; Ji et al., 2014) and brewery effluent (Mata et al., 2014) have also been extensively studied for microalgal cultivation. Gentili et al. (2014) have discussed the possibility of mixing different types of wastewaters for production of algal biomass. However, the use of pulp and paper effluent for the cultivation of microalgae is least explored (Tarlan et al., 2002; Gentili, 2014). The pulp and paper industry is one of the largest producers of plant-based wastewater in the world. About 10- 50 m3 of water is required to produce a tonne of paper. In this scenario, a large amount of wastewater is generated which
has to be treated before its release. The conventional treatment of pulp and paper effluent requires the addition of nutrients as the effluent is limited in nitrogen and phosphorous (Slade et al., 2004). In the present study utilisation of primary treated pulp and paper effluent for the cultivation of a mixed culture of microalgae containing two Scenedesmus sps. was investigated. 2.0 Materials and Methods 2.1 Microalgal culture A mixed culture of microalgae containing two Scenedesmus sp. isolated by Vidyashankar et al. (2103) was used in this study. Two species were initially cultivated individually in Bold Basal Media (Vidyashankar et al., 2013) under controlled conditions at 25oC, 250 µ mol m2 s-1 light intensity under 16:8 light/ dark cycle. 2.2 Wastewater The primary treated wastewater was procured from M/s South Indian Paper Mill located in Mysore, Karnataka, India. The wastewater was carried to the laboratory and was stored in tightly capped glass carboys in cold storage (4oC) for further use. The physico-chemical parameters of wastewater were analysed as per the standard methods (APHA, 1998). For batch studies to assess the effect of wastewater on microalgal growth, the wastewater was diluted with distilled water to obtain the required concentration (5%, 10%, 20%, 40%, 60%, 80%, and 100%). 2.3 Experimental set up The microalgal cultivation studies were conducted in outdoor open circular ponds (1m x 1.5m) of 30L capacity for 28 days. The intermittent manual mixing was provided to avoid the sedimentation of microalgae. During the study, the ambient temperature and the light
intensity were observed to be 23±3 oC and 0.8- 1.8 Kilo lux. The light intensity was measured using digital lux metre (TES 1332, TES Electrical Electronic Corp, Taiwan). Samples were withdrawn at regular intervals and was analysed for NO3-N, PO4-P, iron, zinc, Chemical oxygen demand (COD), biological oxygen demand (BOD) and total organic carbon (TOC). COD, BOD, nitrate and phosphate analysis was carried out as per the standard methods (APHA, 1998). Iron and zinc content was analysed using Atomic Absorption Spectroscopy (AAS). TOC was analysed by TOC analyser. All the experiments were carried out in duplicates. Once the microalgal cultures reached the stationary phase, they were harvested by centrifugation and the obtained biomass were sundried. The lipids from dried biomass were extracted (Moisture content- 6% (w/w)) with hexane using Soxhlet apparatus (Sarat Chandra et al., 2016). Further, Fatty Acid Methyl Ester (FAME) analysis was conducted as per the protocol described in Sarat Chandra et al. (2016) 2.4 Determination of microalgal growth The microalgal growth was determined by measuring the optical density (OD) of culture at 560 nm using a UV/ Visible spectrophotometer (Model: UV/1800A, Shimadzu Corporation, Kyoto, Japan). A relationship between optical density and biomass concentration (g L-1) was established using a calibration curve and it was found that OD560 value of 1 was equivalent to 0.7 g L-1 of biomass. 3.0 Results and Discussion 3.1 Wastewater characterisation The wastewater was characterised for its physico-chemical properties. The pH, conductivity and alkalinity of wastewater were 5.41±0.56, 3932±10 µs cm-1 and 228±4.29 mg L-1. The Total Solids (TS) content of the wastewater was 6446±23.24 mg L-1 with 3580±10.23 mg L-
1
of Total Dissolved Solids (TDS), and 2866±16.56 mg L-1 of Total Suspended Solids (TSS).
The COD, BOD and TOC of the wastewater were 3000.15±28.15 mg L-1, 2944±12.34 mg L-1 and 198±8 mg L-1 respectively. The nitrogen, phosphorous and sulphur are the major nutrients required for the growth of microalgae. A C: N: P ratio of 46.1: 7.7: 1 has been reported as optimal for the growth of microalgae (Hillebrand and Sommer, 1999). The N: P ratio of the paper and pulp wastewater in the present study was 1:3 with the total nitrate being 9.932±1.87 mg L-1 and phosphate being 30.25±3.28 mg L-1. The sulphates (86.48±2.56 mg L-1) and other media constituents such as iron (9.729±0.47 mg L-1) and zinc (7.23±0.38 mg L1
) were found to be sufficient to support the growth of algae. The wastewater also showed a
chloride content of 204±5.68 mg L-1. 3.2 Indoor laboratory studies The indoor microalgal growth studies were conducted in laboratory using 250ml Erlenmeyer flasks. The microalgae were cultivated with various concentrations of wastewater (5%, 10%, 20%, 40%, 60%, 80% and 100%). The microalgal growth was recorded at regular intervals in terms of OD560. The growth profile of microalgal culture at various concentrations of wastewater is shown in Figure 1a. It is evident from Figure 1a that with the increase in the concentration of wastewater (upto 60%), the microalgal growth also increased and was observed to be better than control cultures grown in Bold Basal Medium. However, the microalgal growth was marginally inhibited at wastewater concentrations of > 60% . Therefore, the 60% wastewater (60:40 dilution of wastewater with water) was considered as optimum for microalgal cultivation. 3.3 Wastewater treatment by mixed microalgal culture in outdoor open pond Based on the indoor laboratory studies, 60% concentration of wastewater was chosen as optimum and further studies were conducted in outdoor open ponds. The outdoor open pond cultivation study was carried out for 28 days in a batch mode. The pH of the culture was
monitored on a regular basis throughout the study period and was in the range of 8-10. The biomass was partly harvested when the cultures reached the stationary phase on 16th day and wastewater was recycled. The biomass was harvested at the end of the study period on 28th day. The growth profile of the mixed microalgal culture in outdoor open pond is given in Figure. 1b. The drop in the OD on the 16th day was due to the harvesting of biomass. The study suggests that paper and pulp wastewater could support the long duration microalgal cultivation. 3.4 Nutrient removal Microalgae have the potential for the removal of nutrients from wastewater and cultivation of microalgae in wastewater is a cost effective approach for remediation of organic load and nutrients. The efficiency of the effluent treatment by microalgae is related to its ability to reduce the COD, BOD, nitrogen and phosphorous levels in the effluent. The profile of COD, TOC and BOD removal is shown in Figure 2a. Microalgal cultivation in the effluent resulted in 89% and 75% removal of COD and BOD respectively. Nitrogen and phosphorous are the major nutrients required for microalgal growth, as both nitrogen and phosphorous found in nucleic acids, proteins, carbohydrates and the intermediates of carbohydrate metabolism. The removal of nitrate and phosphate was analysed by periodic determination of NO3-N and PO4P levels in the wastewater used for the cultivation of microalgae in outdoor open pond. The profile of NO3-N and PO4-P removal from wastewater by microalgae is showed in the Figure 2b. On 28th day of microalgal cultivation, 65% and 71.29% removal of NO3-N and PO4-P, respectively, was achieved. Iron and zinc are the micronutrients required for the growth of the microalgae and at the end of 28 days of microalgal cultivation, 25% and 68% removal of iron and zinc respectively from wastewater was recorded (Figure. 2c). Hongyang et al. (2011) has reported 77.8%, 88.8% and 70.3% COD, nitrogen and phosphorus removal respectively from soybean processing wastewater via cultivation of microalga Chlorella pyrenoidosa. An 88% COD removal rate of piggery wastewater by Chlorella sps has been
reported by Travieso et al. (2006) for an initial COD of 250 mg L-1 at 190 hrs which is much higher than obtained in the present study (34% COD reduction for an initial COD of 1520 mg L-1 in 190 hrs). This may be attributed to the difference in microalgal species and the wastewater considered. 3.5 Biomass characterization Microalgal biomass was harvested from outdoor open pond at the end of the cultivation period of 28 days and analysed for its biochemical composition. The microalgal biomass was found to be rich in protein (16±2.45%), iron (2.48 mg g-1) and zinc (0.366 mg g-1). The carbohydrate and lipid content (% wt/wt) of the biomass were 18.90±3.2% and 15.8±4% respectively. The biomass was also analysed for its fatty acid composition (Figure 3). The palmitic acid (C16:0), alpha linolenic acid (C 18:3) and oleic acid (C 18:2) were the major fatty acids contributing upto 32%, 24% and 18.52 respectively in the biomass harvested on 16th day. The alpha linolenic acid, palmitic acid, oleic acid and linoleic acid were major fatty acids contributing upto 31%, 29%, 13% and 13% respectively in the biomass harvested on 28th day. Therefore, the study suggests that the microalgal biomass thus produced utilising the paper and pulp wastewater has the potential for food, feed applications. 4.0 Conclusion Microalgae for removal of nutrients and organic load from pulp and paper mill effluent was studied. A removal of upto 89% and 75% of COD and BOD respectively was achieved. NO3-N and PO4-P removal of upto 65% and 71.29% respectively was observed at the end of 28 days. The biomass obtained was found to be rich in protein and also showed significant presence of α-linolenic acid. Therefore, the present study showed that microalgae can be effectively used for removal of nutrients and organic matter from paper and pulp mill wastewater with concomitant production of microalgal biomass for value added application.
Acknowledgements Financial support (BT/PR1311/PBD/26/267/2011) from Department of Biotechnology (DBT), Government of India is gratefully acknowledged. TSC is thankful to Council of Scientific and Industrial Research (CSIR) for providing research fellowship. The authors thank Director, CSIR-CFTRI for encouragement. References 1) American Public Health Association (APHA), 1998. Standard methods for the examination of water and wastewater, 20th ed. APHA AWWA WPCF, Washington, DC. 2) Beevi, U. S., Sukumaran, R. K., 2014. Cultivation of microalgae in dairy effluent for oil production and removal of organic pollution load. Bioresource Technology. 165, 295301. 3) Cho, S., Lee, N., Park, S., Yu, J., Luong, T. T., Oh, Y., Lee, T., 2013. Microalgae cultivation for bioenergy production using wastewaters from a municipal WWTP as nutritional sources. Bioresource Technology. 131, 515- 520. 4) Gentili, F. G., 2014. Microalgal biomass and lipid production in mixed municipal, dairy, pulp and paper wastewater together with added flue gases. Bioresource Technology. 169, 27- 32. 5) Hillebrand, H., Sommer, U., 1999. The nutrient stoichiometry of benthic microalgal growth: redfield proportions are optimal. Limnology and Oceanography 44, 440- 446. 6) Hongyang, Su., Yalei, Z., Chunmin, Z., Xuefei, Z., Jinpeng, L., 2011. Cultivation of Chlorella pyrenoidosa in soybean processing wastewater. Bioresource Technology. 102(21), 9884-9890. 7) Ji, F., Liu, Y., Hao, R., Li, G., Zhou, Y., Dong, R., 2014. Biomass production and nutrients removal by a new microalgae strain Desmodesmus sp. in anaerobic digestion wastewater. Bioresource Technology. 161, 200- 207.
8) Ji, M.K., Kabra, A. N., Salama, E.S., Roh, H.S., Kim, J. R., Jeon, B., Lee, D. S., 2014. Effect of mine wastewater on nutrient removal and lipid production by a green microalga Micratinium reisseri from concentrated municipal wastewater. Bioresource Technology. 157, 84- 90. 9) Komolafe, O., Orta, S. B. V., Monje-Ramirez, I., Noguez, I. Y., Harvey, A. P., Ledesma, M. T. O., 2014. Biodiesel production from indigenous microalgae grown in wastewater. Bioresource Technology. 154, 297- 304.
10) Li, Y., Chen, Y.-F., Chen, P., Min, M., Zhou, W., Blanca, M., 2011. Characterization of a microalga Chlorella sp. well adapted to highly concentrated municipal wastewater for nutrient removal and biodiesel production. Bioresource Technology. 102, 5138–5144. 11) Mata, T. M., Mendes, A. M., Caetano, N. S., Martins, A. A., 2014. Sustainability and economic evaluation of microalgae grown in brewery wastewater. Bioresource Technology. 168, 151-158. 12) Ramanna, L., Guldhe, A., Rawat, I., Bux, F., 2014. The optimization of biomass and lipid yields of Chlorella sorokiniana when using wastewater supplemented with different nitrogen sources. Bioresource Technology. 168, 127- 135.. 13) Sarat Chandra, T., Deepak, R. S., Maneesh Kumar, M., Mukherji, S., Chauhan, V. S., Sarada, R., Mudliar, S. N., 2016. Evaluation of indigenous fresh water microalga Scenedesmus obtusus for feed and fuel applications: Effect of Carbon dioxide, light and nutrient sources on growth and biochemical characteristics. Bioresource Technology. 207, 430-439. 14) Slade, A.H., Ellis, R.J., vanden Heuvel, M., Stuthridge, T.R., 2004. Nutrient minimisation in the pulp and paper industry: an overview. Water Science and Technology. 50, 111– 122 15) Tarlan, E., Dilek, F.B., Yetis, U., 2002. Effectiveness of algae in the treatment of a woodbased pulp and paper industry wastewater. Bioresource Technology. 84, 1–5
16) Travieso, L., Benitez, F., Sanchez, E., Borja, R., Martin, A., Colmenarejo, M. F., 2006. Batch mixed culture of Chlorella vulgaris using settled and diluted piggery waste. Ecological Engineering. 28(2), 158-165. 17) Uggetti, E., Sialve, B., Latrille, E., Steyer, J., 2014. Anaerobic digestate as substrate for microalgae culture: The role of ammonium concentration on the microalgae productivity. Bioresource Technology. 152, 437-443.
18) Vidyashankar, S., Deviprasad, K., Chauhan, V. S., Ravishankar, G. A., Sarada, R., 2013. Selection and evaluation of CO2 tolerant indigenous microalga Scenedesmus dimorphus for unsaturated fatty acid rich lipid production under different culture conditions. Bioresource Technology. 144, 28–37. 19) Wu, P., Teng, J., Lin, Y., Hwang, S, J., 2013. Increasing algal biofuel production using Nannocholropsis oculata cultivated with anaerobically and aerobically treated swine wastewater. Bioresource Technology. 133, 102- 108. 20) Yang, J., Xu, M., Zhang, X., Hu, Q., Sommerfeld, M., Chen, Y., 2011. Life-cycle analysis on biodiesel production from microalgae: Water foot print and nutrient balance. Bioresource Technology. 102, 159-165.
Figure Captions Figure 1 (a) Growth profile of mixed microalgal culture at various concentrations of pulp and paper mill effluent in laboratory, and (b) Growth profile of mixed microalgal culture in outdoor open pond with 60% pulp and paper mill effluent. Figure 2 Profile of (a) COD, BOD and TOC removal (b) Nitrate and phosphate removal and (c) iron and zinc removal from pulp and paper mill effluent utilised for cultivation of microalgae in outdoor open pond. Figure 3 Fatty acid profile of mixed microalgal culture cultivated with pulp and paper mill effluent.
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Highlights
COD and BOD removal of upto 89% and 75% respectively was achieved. NO3-N and PO4-P removal of upto 65% and 71.29% respectively was observed. An enhanced growth of microalgae was observed with 60% effluent. The palmitic acid and α-linolenic acid were the major fatty acids of microalgae.
Keywords: pulp and paper mill effleunt, Scenedesmus, COD, Lipids, nutrient removal
Abstract: A mixed culture of microalgae, containing two Scenedesmus species, was analysed to determine its potential in coupling of pulp and paper mill effluent treatment and microalgal cultivation. Laboratory studies suggested that 60% concentration of wastewater resulted in better biomass growth than the control. A maximum of 89% and 75% removal of COD and BOD respectively was achieved with microalgal cultivation in outdoor open pond. By the end of the cultivation period, 65% removal of NO3-N and 71.29% removal of PO4-P was observed. The fatty acid composition of mixed microalgal culture cultivated with effluent showed the palmitic acid, oleic acid, linoleic acid and α-linolenic acid as major fatty acids. The results obtained suggest that pulp and paper mill effluent could be used effectively for cultivation of microalgae to minimise the freshwater and nutrient requirements.