Bioresource Technology 169 (2014) 27–32
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Microalgal biomass and lipid production in mixed municipal, dairy, pulp and paper wastewater together with added flue gases Francesco G. Gentili ⇑ Department of Wildlife, Fish and Environmental Studies, Swedish University of Agricultural Sciences, 901 83 Umeå, Sweden
h i g h l i g h t s Growth of microalgae on mixed municipal and industrial wastewater. High nitrogen and phosphorus removal was achieved in all treatments. Selenastrum minutum had the highest biomass and lipids yields. Lipid content was negatively correlated to the nitrogen concentration. Mixtures of wastewater have great potential to produce algal biomass and lipid.
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Article history: Received 1 April 2014 Received in revised form 17 June 2014 Accepted 18 June 2014 Available online 26 June 2014 Keywords: Algae Nitrogen removal Phosphorus removal Total lipids Wastewater
a b s t r a c t The aim of the study was to grow microalgae on mixed municipal and industrial wastewater to simultaneously treat the wastewater and produce biomass and lipids. All algal strains grew in all wastewater mixtures; however, Selenastrum minutum had the highest biomass and lipids yields, up to 37% of the dry matter. Nitrogen and phosphorus removal were high and followed a similar trend in all three strains. Ammonium was reduced from 96% to 99%; this reduction was due to algal growth and not to stripping to the atmosphere, as confirmed by the amount of nitrogen in the dry algal biomass. Phosphate was reduced from 91% to 99%. In all strains used the lipid content was negatively correlated to the nitrogen concentration in the algal biomass. Mixtures of pulp and paper wastewater with municipal and dairy wastewater have great potential to grow algae for biomass and lipid production together with effective wastewater treatment. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The reclamation of wastewater, both municipal and industrial, is of pivotal importance to achieving sustainability in our society at the global level. Often in traditional and well established wastewater treatment techniques the reduction of nitrogen and phosphorus is energy demanding. On the one hand, traditional treatments efficiently reduce the concentration of N and P; on the other, the treatments waste these important and vital nutrients through denitrification or deposition in landfill. It has been estimated that nitrogen pollution alone costs the European Union between €70 billion and €320 billion per year (Sutton et al., 2011). Furthermore, the energy required to produce N and P fertilizers is high, 11.1 and 10 kWh/kg, respectively (Olsson, 2012).
⇑ Tel.: +46 90 7868196. E-mail address: [email protected] http://dx.doi.org/10.1016/j.biortech.2014.06.061 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Phosphorus resources are limited and we are rapidly approaching production peak (Cordell et al., 2009); hence, recycling this vital element is a pivotal challenge of the 21st century. Wastewater reclamation together with nutrient recycling is crucial to achieving environmental sustainability. The pulp and paper industry is the world’s largest producer of plant-based wastewater (Reid et al., 2008). Even though the amount of water per ton of paper produced has been decreasing over time, between 10 and 50 m3 of water are still needed to produce a ton of paper (Pizzichini et al., 2005; Buyukkamaci and Koken, 2010). Consequently, at the global scale the pulp and paper industries produce a great amount of wastewater that has to be treated before being released into the environment. Even though the wastewater from the pulp and paper industry is rich in carbon it is limited in nitrogen and phosphorus. Hence in conventional wastewater treatment processes in the pulp and paper industry nutrients addition has been carried out to ensure microorganisms
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F.G. Gentili / Bioresource Technology 169 (2014) 27–32
growth for treating the wastewater (Thompson et al., 2001; Slade et al., 2004). Algae have been successfully used to remove of COD (chemical oxygen demands), colour and organic xenobiotics from diluted pulp and paper wastewater, with nutrients added to support the algal growth (Tarlan et al., 2002). The wastewater produced by the dairy industry is rich in nitrogen and phosphorus (Kothari et al., 2012). Generally the dairy industry produces a volume of wastewater ca 2.5 times the volume of the milk processed, resulting in large amounts of wastewater (Ramasamy et al., 2004) and sludge. In a study dealing with agroindustrial wastewater from dairy and pig farming algal growth could efficiently remove ammonia and phosphorus (González et al., 1997). Municipal wastewater is globally produced in huge amounts and conventional wastewater treatment is costly and energy demanding (Lundin et al., 2000). Urban wastewater is rich in nutrients such as nitrogen and phosphorus (Ruiz-Marin et al., 2010; Doria et al., 2012). The use of algae for wastewater reclamation is of great interest. Algae were already being tested in the treatment of municipal wastewater during the 1950s (Oswald and Gotaas, 1957). However, more recently a considerable amount of work has focused on algal treatment of municipal wastewater (Hoffmann, 1998; Park et al., 2011; Pittman et al., 2011). In some cases reductions in nitrogen and phosphorus of up to 90–95% were achieved (Hoffmann, 1998; Ruiz-Marin et al., 2010). The potential of microalgae to remove N and P during tertiary sewage treatment has already been extensively assessed (Pittman et al., 2011). Furthermore it has been shown that algae grown on wastewater yielded more biomass when additional CO2 was bubbled into the algae culture (Craggs et al., 2012). Even though the interest in using algae to reclaim both municipal and industrial wastewater is continuing to increase, very few if any studies have focused on mixtures of municipal and industrial wastewater, including pulp and paper and dairy. The aims of the present work are the following: (1) the use of microalgae to reclaim mixed municipal and industrial wastewater or sludge together with CO2 addition; (2) to investigate how mixtures of municipal and industrial wastewater or sludge could provide good substrates for algal growth without the need for dilution with clean fresh water or nutrients supplementation; (3) to quantify biomass and lipid yield of three algal strains grown on three wastewater mixtures. 2. Methods 2.1. Local strain isolation Wastewater samples from the local municipal wastewater treatment plant (Umeva, Umeå northern Sweden 63°520 N) were placed in a closed bottle near the laboratory window under continuous agitation by a magnetic stirrer. After a few weeks of incubation, algal growth was visible to the naked eye and then small aliquots were streaked on sterile agar plates with Bristol medium (Wang and Lan, 2011). Subsequently a single colony was transferred to a new sterile plate with Bristol medium to obtain an algal monoculture, albeit it was not axenic. 2.2. Morphology of the algae Light microscopy was used to aid in identifying the local isolated algal strain in accordance with Bellinger and Sigee (2010). The strain isolated as described above and the strains purchased from the UTEX algal collection were observed under a light microscope (Optika B-353 LD2, Optika, Italy).
2.3. Growing conditions Municipal influent wastewater was collected from the local wastewater treatment plant (Umeva, Umeå, Sweden) and pulp and paper influent was collected from SCA Obbola (Obbola, Sweden), which uses chemical pulping (sulphate process), while dairy final effluent and sludge was collected from the local dairy (Norrmejerier, Umeå). The following wastewater mixtures were prepared: (a) pulp and paper influent 4:1 dairy sludge; (b) pulp and paper influent 1:1 municipal influent; (c) pulp and paper influent 2:1 dairy final effluent. All the mixtures were left to settle in the coldroom over night before the supernatant was transferred into tubes. Sterile 50 ml plastic tubes each received 39 ml of wastewater mixture. Two microalgal strains, Scenedesmus dimorphus (417) and Selenastrum minutum (326) were purchased from UTEX, The Culture Collection of Algae at the University of Texas at Austin (in parenthesis is the strains UTEX id); while a third strain was isolated locally (see above) and identified as Scenedesmus sp. The three algal strains were grown for a couple of weeks on filtered and autoclaved municipal wastewater influent; then they were harvested by centrifugation at 3580g for 5 min, the supernatant was discarded and the pelleted cells were resuspended in tap water. Each tube containing 39 ml of wastewater mixture was inoculated with 1 ml of algal culture at a concentration of 0.1 g/l dry weight. The control tubes received only the wastewater mixtures without algal addition. The controls were coved by aluminium foil to avoid the growth of native algae. The experiment was carried out at the local combined heat and power plant (Umeå Energi, Umeå), where the samples were continuously illuminated with fluorescent lamps at a PAR (photosynthetically active radiation) of 130 lE m2 s1. The temperature was recorded every 5 min and ranged from 21.7 to 32.2 °C with a mean value of 27.5 °C. The flue gases from the combined heat and power plant, having approximately a 10% CO2 concentration, were bubbled at a flow rate of 50 ml min1. The flue gases were bubbled into the tubes using a sterile plastic pipette with a volume of 1 ml. The experiment was ended 6 days after inoculation. 2.4. Chemical analyses, biomass quantification and lipid extraction The pH of the wastewater mixtures was measured at the start of the experiment and again at the end of the experiment for all the samples. A Beckman 295 pH meter (Beckman Coulter, USA) with a Red Rod pH electrode (Radiometer Analytical, France) was used. At the beginning of the experiment total COD analyses were performed in duplicate samples of each non-inoculated noncentrifuged mixture (APHA, 1998). Furthermore, at the beginning of the experiment, duplicate samples of each non-inoculated wastewater mixture were centrifuged at 3580g for 5 min. Then the supernatant was analysed using a spectrophotometer (DR 3900 Hach Lange, Germany) following the manufacturer instructions (Hach Lange, Germany); while the pellets were transferred to a dry pre-weighed Eppendorf tube and dried at 70 °C for 24 h. The same procedure was performed at the end of the experiment 6 days after algal inoculation. The algal removal of COD, NO3, NH4, PO4, total N and total P in the different wastewater mixtures was calculated as follows: [(initial value final value)/initial value] * 100. Total N removal was followed through the entire experiment by collecting two samples every 2 days for each treatment. Algal biomass production was calculated by subtracting the initial total suspended solids of the wastewater mixtures (control at the beginning) and the algal inoculum from the final harvested biomass. All weight measurements were done with a high precision balance (Kern ABT 120-5DM; readout 0.01 mg; Kern, Germany). Nitrogen quantification analyses of the dry biomass at final harvest
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were performed by an Elemental Analyser–Isotope Ratio Mass Spectrometer (EA–IRMS) at the Department of Forest Ecology and Management, Swedish University of Agricultural Sciences (SLU) Umeå, Sweden. The dry harvested biomass was milled in a ball mill (Retsch MM 200, Retsch Germany) and then the lipids were extracted from all inoculated samples by a simplified Folch method (Axelsson and Gentili, 2014) with two extractions. Prior to the extractions, a quantity of deionised water similar to the amount of intracellular water was added to each sample of dry milled algae biomass in order to improve the extraction. 3. Results and discussion 3.1. Strain identification The isolated algal strain from local municipal wastewater was identified as Scenedesmus sp. This confirmed that algae belonging to the genus Scenedesmus are frequent in municipal wastewaters, even from different geographical locations (Doria et al., 2012; Abdel-Raouf et al., 2012). 3.2. Biomass and lipids production The three algal strains grew in all the wastewater mixtures used and the biomass yields were due to algae growth and not other microorganisms, as clearly indicated by the very small changes in the controls (Table 1). Interestingly, S. minutum had both the highest biomass and the highest lipid concentration compared to the other two strains used in all the wastewater mixtures (Tables 1 and 3). The algae grown on wastewater mixtures (b) and (c) had higher lipid content than the algae grown on wastewater mixture (a) (Table 3). These results could be due to the lower nutrients content of wastewater mixtures (b) and (c) compared to wastewater mixture (a) (Table 2). It is possible that the algal strains growing in wastewater (b) and (c) used up all the available nitrogen after a few days and then became nitrogen limited, triggering lipid accumulation. This was confirmed by the fact that total nitrogen was reduced already after 2 days from the start of the experiment (see below). The UTEX strains S. dimorphus and S. minutum had a higher biomass yield than the local isolate in all the wastewater mixtures (Table 1). One explanation could be that the local strain, isolated from municipal wastewater having a mean year temperature of 13.5 °C (ranging from 9 to 20 °C), was more susceptible to a relatively high temperature (mean value 27.5 °C) than the two UTEX strains. However, it was relevant to perform the experiment at this temperature because the pulp and paper industry produces and releases large quantities of wastewater having temperatures in the range of 27–29 °C. The lipid concentration of S. dimorphus was higher than in other Scenedesmus grown on municipal wastewater or artificial media (Rodolfi et al., 2009; Doria et al., 2012). In a previous study, lipid concentration of the same algal strains S. dimorphus and S. minutum grown on municipal influent was 14% and 17% respectively of the biomass dry weight (Axelsson and Gentili, 2014). In a study using another strain of S. dimorphus
biomass yield and lipid concentration were respectively similar and higher (30.7%) compared to the results obtained in the present study. However in that study S. dimorphus was grown for longer time (17 days) on an artificial medium with the addition of different sources of nitrogen (Shen et al., 2009). The highest algae biomass production was achieved in wastewater mixture (a), which also had the greatest nitrogen and phosphorus concentration at the beginning of the experiment (Table 2). In this wastewater mixture the green microalgal strain S. minutum had the highest biomass yield (Table 1). Regression analyses in all the strains used showed a negative and significant (p < 0.05) correlation between nitrogen and lipid concentration in the algae dry biomass. Their lipid contents suggest the strains used have good potential as sources of biofuel when grown in the mixtures of wastewater used in the present study. This aspect is important from an environmental as well as from an economic point of view.
3.3. Nutrient removal and nitrogen concentration of the algae biomass On one hand, in all the three algal strains grown in wastewater mixture (b) and (c) and for the local strain grown in wastewater mixture (a), total nitrogen was reduced after two days at the level (data not shown) of the final harvest. On the other hand, in wastewater mixture (a) S. dimorphus and S. minutum showed a slightly higher total nitrogen concentration than at the final harvest (data not shown). In a previous study using wastewater supernatant from the secondary settler, autochthonous species of Scenedesmus could reduce ammonium nitrogen up to 99.9% after 24 h of growth in a photobioreactor (Di Termini et al., 2011). In another study using primary settled municipal wastewater the Chlorella vulgaris could reduce ammonium-nitrogen and total nitrogen by 74% and 69.1% respectively after 6 days of growth and it took 10 days (Lau et al., 1995) to reach a similar reduction obtained in the present study. At the end of the experiment in wastewater mixture (a) S. minutum had a slightly lower total N removal than the other two algal strains (Fig. 1). Total P removal was slightly higher and lower compared to the local isolate and S. dimorphus, respectively (Fig. 1). Even though the initial level of nitrate was low, approximately 1 mg/l (Table 2), S. minutum had a smaller reduction than the other two algal strains (Fig. 1). S. dimorphus showed the highest reduction in total P and PO4, while total N was reduced to a very similar extent as that achieved by the local isolate (Fig. 1). In another study using agroindustrial wastewater, S. dimorphus showed an ammonia removal of 95% (González et al., 1997), very similar to the removal obtained in the present study (99%, Figs. 1–3). In all the wastewater mixtures the biomass of the local isolate had a higher nitrogen concentration than those of the other two strains (Table 4). This explains why the local strain exhibited a total nitrogen removal very similar to that of the other two strains even though its yield of biomass was less (Figs. 1–3 and Table 1). On the one hand a higher nitrogen concentration in the biomass is a positive feature in cases where it may be used as a biofertilizer; on the other hand, it is negative with regard to biogas production, where a high nitrogen content in the algal biomass can inhibit biogas production
Table 1 Biomass in the controls, uninoculated wastewater mixtures, at the beginning and end of the experiment and algal biomass of the three strains in the three different wastewater mixtures at the end of the experiment. Values expressed in g/l are the mean ± SE of three replicates. Wastewater
Control at start
Control at the end
Local isolate
S. dimorphus
S. minutum
a b c
0.31 ± 0.01 0.02 ± 0.002 0.02 ± 0
0.35 ± 0.01 0.08 ± 0.01 0.07 ± 0.01
0.64 ± 0.04 0.62 ± 0.01 0.7 ± 0.11
1.24 ± 0.03 0.92 ± 0.12 0.86 ± 0.18
1.49 ± 0.09 0.83 ± 0.09 1.12 ± 0.02
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Table 2 Nutrients, total COD, supernatant COD and pH at the beginning of the experiment in the wastewater mixtures. Values expressed in mg/l are the mean ± SE of two replicates. WW mixture
NO3-N mg/l
NH4-N mg/l
Tot N mg/l
PO4-P mg/l
Tot P mg/l
COD tot
COD S
pH
A B c
1.06 ± 0.01 1.08 ± 0.05 1.25 ± 0.06
22.35 ± 0.25 14.75 ± 0.05 21 ± 0.4
44.65 ± 2.05 22.65 ± 2.15 25.1 ± 0.3
10.1 ± 0.1 1.6 ± 0.005 2.99 ± 0.02
12.75 ± 0.05 1.74 ± 0.08 3.18 ± 0.00
1905 ± 25 1325 ± 0 1167 ± 22
278 ± 1 167 ± 3 224 ± 2.5
7.37 7.48 7.73
Table 3 Lipids extracted as % of sample dry weight of three algal strains grown on three different wastewater mixtures. Values are the mean ± SE of three replicates. Wastewater
Local isolate
S. dimorphus
S. minutum
a b c
17.4 ± 0.39 28.1 ± 1.1 25.0 ± 4.3
19.5 ± 1.3 24.0 ± 3.9 25.7 ± 1.0
27.9 ± 0.9 37.2 ± 3.0 32.0 ± 0.3
Fig. 3. Nutrient removal by the three strains used expressed as a percentage of the starting concentration in wastewater mixture (c). Bars represent the mean ± SE of three replicates.
Table 4 Nitrogen concentration as % of the harvested biomass dry weight of the three strains in the three different wastewater mixtures. a + C stands for algae biomass nitrogen concentration plus control biomass; while a C stands only algae biomass given by the final biomass minus the control biomass. NA stands for not available.
Fig. 1. Nutrient removal by the three strains used expressed as a percentage of the starting concentration in wastewater mixture (a). Bars represent the mean ± SE of three replicates.
Fig. 2. Nutrient removal by the three strains used expressed as a percentage of the starting concentration in wastewater mixture (b). Bars represent the mean ± SE of three replicates.
Wastewater
Control at the end
Local isolate
S. dimorphus
S. minutum
a+C aC b c
7.68 ± 0.10 – NA NA
5.92 ± 0.14 4.81 ± 0.26 3.63 ± 0.05 3.84 ± 0.67
3.50 ± 0.28 2.32 ± 0.27 2.52 ± 0.55 3.35 ± 0.59
3.05 ± 0.06 1.97 ± 0.09 2.62 ± 0.34 2.49 ± 0.08
(Golueke et al., 1957; Zhong et al., 2012). In general the removal trends for ammonium, total nitrogen, phosphate and total phosphorus were similar for all the strains in all the wastewater mixtures used (Figs. 1–3). Major variations were apparent in nitrate removal (Figs. 1–3); however, nitrate was present in such low concentrations that a small change will greatly influence the overall trend. The nitrogen concentration of the harvested biomass from wastewaters (b) and (c) confirmed that ammonium had been taken up and not stripped into the atmosphere through flue gas bubbling (compare Tables 1, 4 and 2). In the case of wastewater (a), even though the ammonium and total nitrogen present in the supernatant were drastically reduced (Fig. 1), it can be speculated that a portion of the nitrogen contained in the original biomass was released into the solution over time and, consequently, some of the nitrogen was probably stripped away by bubbling the flue gases. In another study it was found that algae growing in municipal effluent bubbled with flue gases had a biomass yield approximately three times higher than algae bubbled with air; the latter had a very large proportion of ammonia stripped into the atmosphere (unpublished results). Ammonia stripping under air bubbling in small bottles has been shown in a previous study (Talbot and de la Noüe, 1993).
F.G. Gentili / Bioresource Technology 169 (2014) 27–32
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2001; Slade et al., 2004). Furthermore, the constantly increasing interest in growing algae will lead to an increased demand for water and nutrients. Hence the present study showed that pulp and paper wastewater mixed with dairy sludge/effluent or municipal wastewater can be used and treated by algae without the addition of artificial fertilizers. 4. Conclusions
Fig. 4. Total COD removal by the three strains used expressed as a percentage of the starting concentration in three wastewater mixtures. Bars represent the mean ± SE of two replicates.
The interest in growing algae as sources of biomass and bioenergy is constantly increasing. The cultivation of algae demands large quantities of water and nutrients. The use of wastewater to grow algae is particularly interesting from the point of view of sustainability. However, not all types of wastewater are suitable for algae cultivation. This study shows how mixing different types of wastewater, both municipal and industrial, makes it possible to produce algae biomass with a high lipid content while at the same time treating the wastewater with added flue gases. Acknowledgements
The pH for all treatments at final harvest ranged from 7.71 to 8.39. Total COD was greatly reduced in wastewater mixture (c) with S. minutum, and in wastewater mixture (a) with the local strain from 67% to 92.7% respectively (Fig. 4). However this great reduction of the total COD is due to the harvesting by centrifugation that remove both the algae and the particles present in the wastewater mixtures. Considering COD in the supernatant, the highest reduction of 49.8% was reached in wastewater mixture (a) with the local strain (Fig. 5). In wastewater mixture (c) all algae had a negative effect on COD of the supernatant (Fig. 5). The algal strain S. minutum had a negative effect on supernatant COD in all three wastewater mixtures (Fig. 5). A negative impact of algal growth on COD has been previously reported in different municipal wastewaters and swine manure (Wang et al., 2010; Min et al., 2014). Wang et al. (2010) suggested that the algae excreted organic compounds in the growing substrate. Considering that in the present study the algae grew in autotrophic regime with continuous flue gases bubbling and light irradiation then organic molecules have been excreted into the liquid culture. In conventional wastewater treatment processes of the pulp and paper industry, nutrients have been added to ensure microorganisms growth for treating the wastewater (Thompson et al.,
Fig. 5. Supernatant COD removal by the three strains used expressed as a percentage of the starting concentration in three wastewater mixtures. Bars represent the mean ± SE of two replicates.
I greatly appreciate the financial support of the Swedish Energy Agency, SP Processum and the Kempe Foundation. I am thankful to Mr. Martin Axelsson, Mrs. Ulla Britt Östman and Mr. Hadi Soroosh for technical help. The availability of the technical staff at Umeå Energi, Umeva, Norrmejerier and SCA Obbola AB was greatly appreciated. References Abdel-Raouf, N., Al-Homaidan, A.A., Ibraheem, I.B.M., 2012. Microalgae and wastewater treatment. Saudi J. Biol. Sci. 19, 257–275. APHA, 1998. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington. Axelsson, M., Gentili, F., 2014. A single-step method for rapid extraction of total lipids from green microalgae. Plos One, e89643. http://dx.doi.org/10.1371/ journal.pone.0089643. Bellinger, E.G., Sigee, D.C., 2010. Freshwater Algae: Identification and Use as Bioindicators. Wiley-Blackwell (p. 271). Buyukkamaci, N., Koken, E., 2010. Economic evaluation of alternative wastewater treatment plant options for pulp and paper industry. Sci. Total Environ. 408, 6070–6078. Cordell, D., Drangert, J.O., White, S., 2009. The story of phosphorus: global food security and food for thought. Global Environ. Change 19, 292–305. Craggs, R., Sutherland, D., Campbell, H.J., 2012. Hectare-scale demonstration of high rate algal ponds for enhanced wastewater treatment and biofuel production. Appl. Phycol. 24, 329–337. Di Termini, I., Pressone, A., Cattaneo, C., Rovatti, M., 2011. On the nitrogen and phosphorus removal in algal photobioreactors. Ecol. Eng. 37, 976–980. Doria, E., Longoni, P., Scibilia, L., Cella, R., Nielsen, E., 2012. Isolation and characterization of a Scenedesmus acutus strain to be used for bioremediation of urban wastewater. J. Appl. Phycol. 24, 375–383. Golueke, C.G., Oswald, W.J., Gotaas, H.B., 1957. Anaerobic digestion of algae. Appl. Environ. Microbiol. 5, 47–55. González, L.E., Cañizares, R.O., Baena, S., 1997. Efficiency of ammonia and phosphorus removal from a Colombian agroindustrial wastewater by the microalgae Chlorella vulgaris and Scenedesmus dimorphus. Bioresour. Technol. 60, 259–262. Hoffmann, J.P., 1998. Wastewater treatment with suspended and nonsuspended algae. J. Phycol. 34, 757–763. Kothari, R., Pathak, V.V., Kumar, V., Singh, D.P., 2012. Experimental study for growth potential of unicellular alga Chlorella pyrenoidosa on dairy waste water: an integrated approach for treatment and biofuel production. Bioresour. Technol. 116, 466–470. Lau, P.S., Tam, N.F.Y., Wong, Y.S., 1995. Effect of algal density on the nutrient removal from primary settled wastewater. Environ. Pollut. 89, 59–66. Lundin, M., Bengtsson, M., Molander, S., 2000. Life cycle assessment of wastewater systems: influence of system boundaries and scale on calculated environmental loads. Environ. Sci. Technol. 34, 180–186. Min, M., Hu, B., Mohr, M.J., Shi, A., Ding, J., Sun, Y., Jiang, Y., Fu, Z., Griffith, R., Hussain, F., Mu, D., Nie, Y., Chen, P., Zhou, W., Ruan, R., 2014. Swine manurebased pilot-scale algal biomass production system for fuel production and wastewater treatment – a case study. Appl. Biochem. Biotechnol. 172, 1390– 1406. Olsson, G., 2012. Water and Energy – Threats and Opportunities. IWA Publications, London, pp. 68–69.
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Microalgal biomass and lipid production in mixed municipal, dairy, pulp and paper wastewater together with added flue gases Francesco G. Gentili ⇑ Department of Wildlife, Fish and Environmental Studies, Swedish University of Agricultural Sciences, 901 83 Umeå, Sweden
h i g h l i g h t s Growth of microalgae on mixed municipal and industrial wastewater. High nitrogen and phosphorus removal was achieved in all treatments. Selenastrum minutum had the highest biomass and lipids yields. Lipid content was negatively correlated to the nitrogen concentration. Mixtures of wastewater have great potential to produce algal biomass and lipid.
a r t i c l e
i n f o
Article history: Received 1 April 2014 Received in revised form 17 June 2014 Accepted 18 June 2014 Available online 26 June 2014 Keywords: Algae Nitrogen removal Phosphorus removal Total lipids Wastewater
a b s t r a c t The aim of the study was to grow microalgae on mixed municipal and industrial wastewater to simultaneously treat the wastewater and produce biomass and lipids. All algal strains grew in all wastewater mixtures; however, Selenastrum minutum had the highest biomass and lipids yields, up to 37% of the dry matter. Nitrogen and phosphorus removal were high and followed a similar trend in all three strains. Ammonium was reduced from 96% to 99%; this reduction was due to algal growth and not to stripping to the atmosphere, as confirmed by the amount of nitrogen in the dry algal biomass. Phosphate was reduced from 91% to 99%. In all strains used the lipid content was negatively correlated to the nitrogen concentration in the algal biomass. Mixtures of pulp and paper wastewater with municipal and dairy wastewater have great potential to grow algae for biomass and lipid production together with effective wastewater treatment. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The reclamation of wastewater, both municipal and industrial, is of pivotal importance to achieving sustainability in our society at the global level. Often in traditional and well established wastewater treatment techniques the reduction of nitrogen and phosphorus is energy demanding. On the one hand, traditional treatments efficiently reduce the concentration of N and P; on the other, the treatments waste these important and vital nutrients through denitrification or deposition in landfill. It has been estimated that nitrogen pollution alone costs the European Union between €70 billion and €320 billion per year (Sutton et al., 2011). Furthermore, the energy required to produce N and P fertilizers is high, 11.1 and 10 kWh/kg, respectively (Olsson, 2012).
⇑ Tel.: +46 90 7868196. E-mail address: [email protected] http://dx.doi.org/10.1016/j.biortech.2014.06.061 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Phosphorus resources are limited and we are rapidly approaching production peak (Cordell et al., 2009); hence, recycling this vital element is a pivotal challenge of the 21st century. Wastewater reclamation together with nutrient recycling is crucial to achieving environmental sustainability. The pulp and paper industry is the world’s largest producer of plant-based wastewater (Reid et al., 2008). Even though the amount of water per ton of paper produced has been decreasing over time, between 10 and 50 m3 of water are still needed to produce a ton of paper (Pizzichini et al., 2005; Buyukkamaci and Koken, 2010). Consequently, at the global scale the pulp and paper industries produce a great amount of wastewater that has to be treated before being released into the environment. Even though the wastewater from the pulp and paper industry is rich in carbon it is limited in nitrogen and phosphorus. Hence in conventional wastewater treatment processes in the pulp and paper industry nutrients addition has been carried out to ensure microorganisms
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growth for treating the wastewater (Thompson et al., 2001; Slade et al., 2004). Algae have been successfully used to remove of COD (chemical oxygen demands), colour and organic xenobiotics from diluted pulp and paper wastewater, with nutrients added to support the algal growth (Tarlan et al., 2002). The wastewater produced by the dairy industry is rich in nitrogen and phosphorus (Kothari et al., 2012). Generally the dairy industry produces a volume of wastewater ca 2.5 times the volume of the milk processed, resulting in large amounts of wastewater (Ramasamy et al., 2004) and sludge. In a study dealing with agroindustrial wastewater from dairy and pig farming algal growth could efficiently remove ammonia and phosphorus (González et al., 1997). Municipal wastewater is globally produced in huge amounts and conventional wastewater treatment is costly and energy demanding (Lundin et al., 2000). Urban wastewater is rich in nutrients such as nitrogen and phosphorus (Ruiz-Marin et al., 2010; Doria et al., 2012). The use of algae for wastewater reclamation is of great interest. Algae were already being tested in the treatment of municipal wastewater during the 1950s (Oswald and Gotaas, 1957). However, more recently a considerable amount of work has focused on algal treatment of municipal wastewater (Hoffmann, 1998; Park et al., 2011; Pittman et al., 2011). In some cases reductions in nitrogen and phosphorus of up to 90–95% were achieved (Hoffmann, 1998; Ruiz-Marin et al., 2010). The potential of microalgae to remove N and P during tertiary sewage treatment has already been extensively assessed (Pittman et al., 2011). Furthermore it has been shown that algae grown on wastewater yielded more biomass when additional CO2 was bubbled into the algae culture (Craggs et al., 2012). Even though the interest in using algae to reclaim both municipal and industrial wastewater is continuing to increase, very few if any studies have focused on mixtures of municipal and industrial wastewater, including pulp and paper and dairy. The aims of the present work are the following: (1) the use of microalgae to reclaim mixed municipal and industrial wastewater or sludge together with CO2 addition; (2) to investigate how mixtures of municipal and industrial wastewater or sludge could provide good substrates for algal growth without the need for dilution with clean fresh water or nutrients supplementation; (3) to quantify biomass and lipid yield of three algal strains grown on three wastewater mixtures. 2. Methods 2.1. Local strain isolation Wastewater samples from the local municipal wastewater treatment plant (Umeva, Umeå northern Sweden 63°520 N) were placed in a closed bottle near the laboratory window under continuous agitation by a magnetic stirrer. After a few weeks of incubation, algal growth was visible to the naked eye and then small aliquots were streaked on sterile agar plates with Bristol medium (Wang and Lan, 2011). Subsequently a single colony was transferred to a new sterile plate with Bristol medium to obtain an algal monoculture, albeit it was not axenic. 2.2. Morphology of the algae Light microscopy was used to aid in identifying the local isolated algal strain in accordance with Bellinger and Sigee (2010). The strain isolated as described above and the strains purchased from the UTEX algal collection were observed under a light microscope (Optika B-353 LD2, Optika, Italy).
2.3. Growing conditions Municipal influent wastewater was collected from the local wastewater treatment plant (Umeva, Umeå, Sweden) and pulp and paper influent was collected from SCA Obbola (Obbola, Sweden), which uses chemical pulping (sulphate process), while dairy final effluent and sludge was collected from the local dairy (Norrmejerier, Umeå). The following wastewater mixtures were prepared: (a) pulp and paper influent 4:1 dairy sludge; (b) pulp and paper influent 1:1 municipal influent; (c) pulp and paper influent 2:1 dairy final effluent. All the mixtures were left to settle in the coldroom over night before the supernatant was transferred into tubes. Sterile 50 ml plastic tubes each received 39 ml of wastewater mixture. Two microalgal strains, Scenedesmus dimorphus (417) and Selenastrum minutum (326) were purchased from UTEX, The Culture Collection of Algae at the University of Texas at Austin (in parenthesis is the strains UTEX id); while a third strain was isolated locally (see above) and identified as Scenedesmus sp. The three algal strains were grown for a couple of weeks on filtered and autoclaved municipal wastewater influent; then they were harvested by centrifugation at 3580g for 5 min, the supernatant was discarded and the pelleted cells were resuspended in tap water. Each tube containing 39 ml of wastewater mixture was inoculated with 1 ml of algal culture at a concentration of 0.1 g/l dry weight. The control tubes received only the wastewater mixtures without algal addition. The controls were coved by aluminium foil to avoid the growth of native algae. The experiment was carried out at the local combined heat and power plant (Umeå Energi, Umeå), where the samples were continuously illuminated with fluorescent lamps at a PAR (photosynthetically active radiation) of 130 lE m2 s1. The temperature was recorded every 5 min and ranged from 21.7 to 32.2 °C with a mean value of 27.5 °C. The flue gases from the combined heat and power plant, having approximately a 10% CO2 concentration, were bubbled at a flow rate of 50 ml min1. The flue gases were bubbled into the tubes using a sterile plastic pipette with a volume of 1 ml. The experiment was ended 6 days after inoculation. 2.4. Chemical analyses, biomass quantification and lipid extraction The pH of the wastewater mixtures was measured at the start of the experiment and again at the end of the experiment for all the samples. A Beckman 295 pH meter (Beckman Coulter, USA) with a Red Rod pH electrode (Radiometer Analytical, France) was used. At the beginning of the experiment total COD analyses were performed in duplicate samples of each non-inoculated noncentrifuged mixture (APHA, 1998). Furthermore, at the beginning of the experiment, duplicate samples of each non-inoculated wastewater mixture were centrifuged at 3580g for 5 min. Then the supernatant was analysed using a spectrophotometer (DR 3900 Hach Lange, Germany) following the manufacturer instructions (Hach Lange, Germany); while the pellets were transferred to a dry pre-weighed Eppendorf tube and dried at 70 °C for 24 h. The same procedure was performed at the end of the experiment 6 days after algal inoculation. The algal removal of COD, NO3, NH4, PO4, total N and total P in the different wastewater mixtures was calculated as follows: [(initial value final value)/initial value] * 100. Total N removal was followed through the entire experiment by collecting two samples every 2 days for each treatment. Algal biomass production was calculated by subtracting the initial total suspended solids of the wastewater mixtures (control at the beginning) and the algal inoculum from the final harvested biomass. All weight measurements were done with a high precision balance (Kern ABT 120-5DM; readout 0.01 mg; Kern, Germany). Nitrogen quantification analyses of the dry biomass at final harvest
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were performed by an Elemental Analyser–Isotope Ratio Mass Spectrometer (EA–IRMS) at the Department of Forest Ecology and Management, Swedish University of Agricultural Sciences (SLU) Umeå, Sweden. The dry harvested biomass was milled in a ball mill (Retsch MM 200, Retsch Germany) and then the lipids were extracted from all inoculated samples by a simplified Folch method (Axelsson and Gentili, 2014) with two extractions. Prior to the extractions, a quantity of deionised water similar to the amount of intracellular water was added to each sample of dry milled algae biomass in order to improve the extraction. 3. Results and discussion 3.1. Strain identification The isolated algal strain from local municipal wastewater was identified as Scenedesmus sp. This confirmed that algae belonging to the genus Scenedesmus are frequent in municipal wastewaters, even from different geographical locations (Doria et al., 2012; Abdel-Raouf et al., 2012). 3.2. Biomass and lipids production The three algal strains grew in all the wastewater mixtures used and the biomass yields were due to algae growth and not other microorganisms, as clearly indicated by the very small changes in the controls (Table 1). Interestingly, S. minutum had both the highest biomass and the highest lipid concentration compared to the other two strains used in all the wastewater mixtures (Tables 1 and 3). The algae grown on wastewater mixtures (b) and (c) had higher lipid content than the algae grown on wastewater mixture (a) (Table 3). These results could be due to the lower nutrients content of wastewater mixtures (b) and (c) compared to wastewater mixture (a) (Table 2). It is possible that the algal strains growing in wastewater (b) and (c) used up all the available nitrogen after a few days and then became nitrogen limited, triggering lipid accumulation. This was confirmed by the fact that total nitrogen was reduced already after 2 days from the start of the experiment (see below). The UTEX strains S. dimorphus and S. minutum had a higher biomass yield than the local isolate in all the wastewater mixtures (Table 1). One explanation could be that the local strain, isolated from municipal wastewater having a mean year temperature of 13.5 °C (ranging from 9 to 20 °C), was more susceptible to a relatively high temperature (mean value 27.5 °C) than the two UTEX strains. However, it was relevant to perform the experiment at this temperature because the pulp and paper industry produces and releases large quantities of wastewater having temperatures in the range of 27–29 °C. The lipid concentration of S. dimorphus was higher than in other Scenedesmus grown on municipal wastewater or artificial media (Rodolfi et al., 2009; Doria et al., 2012). In a previous study, lipid concentration of the same algal strains S. dimorphus and S. minutum grown on municipal influent was 14% and 17% respectively of the biomass dry weight (Axelsson and Gentili, 2014). In a study using another strain of S. dimorphus
biomass yield and lipid concentration were respectively similar and higher (30.7%) compared to the results obtained in the present study. However in that study S. dimorphus was grown for longer time (17 days) on an artificial medium with the addition of different sources of nitrogen (Shen et al., 2009). The highest algae biomass production was achieved in wastewater mixture (a), which also had the greatest nitrogen and phosphorus concentration at the beginning of the experiment (Table 2). In this wastewater mixture the green microalgal strain S. minutum had the highest biomass yield (Table 1). Regression analyses in all the strains used showed a negative and significant (p < 0.05) correlation between nitrogen and lipid concentration in the algae dry biomass. Their lipid contents suggest the strains used have good potential as sources of biofuel when grown in the mixtures of wastewater used in the present study. This aspect is important from an environmental as well as from an economic point of view.
3.3. Nutrient removal and nitrogen concentration of the algae biomass On one hand, in all the three algal strains grown in wastewater mixture (b) and (c) and for the local strain grown in wastewater mixture (a), total nitrogen was reduced after two days at the level (data not shown) of the final harvest. On the other hand, in wastewater mixture (a) S. dimorphus and S. minutum showed a slightly higher total nitrogen concentration than at the final harvest (data not shown). In a previous study using wastewater supernatant from the secondary settler, autochthonous species of Scenedesmus could reduce ammonium nitrogen up to 99.9% after 24 h of growth in a photobioreactor (Di Termini et al., 2011). In another study using primary settled municipal wastewater the Chlorella vulgaris could reduce ammonium-nitrogen and total nitrogen by 74% and 69.1% respectively after 6 days of growth and it took 10 days (Lau et al., 1995) to reach a similar reduction obtained in the present study. At the end of the experiment in wastewater mixture (a) S. minutum had a slightly lower total N removal than the other two algal strains (Fig. 1). Total P removal was slightly higher and lower compared to the local isolate and S. dimorphus, respectively (Fig. 1). Even though the initial level of nitrate was low, approximately 1 mg/l (Table 2), S. minutum had a smaller reduction than the other two algal strains (Fig. 1). S. dimorphus showed the highest reduction in total P and PO4, while total N was reduced to a very similar extent as that achieved by the local isolate (Fig. 1). In another study using agroindustrial wastewater, S. dimorphus showed an ammonia removal of 95% (González et al., 1997), very similar to the removal obtained in the present study (99%, Figs. 1–3). In all the wastewater mixtures the biomass of the local isolate had a higher nitrogen concentration than those of the other two strains (Table 4). This explains why the local strain exhibited a total nitrogen removal very similar to that of the other two strains even though its yield of biomass was less (Figs. 1–3 and Table 1). On the one hand a higher nitrogen concentration in the biomass is a positive feature in cases where it may be used as a biofertilizer; on the other hand, it is negative with regard to biogas production, where a high nitrogen content in the algal biomass can inhibit biogas production
Table 1 Biomass in the controls, uninoculated wastewater mixtures, at the beginning and end of the experiment and algal biomass of the three strains in the three different wastewater mixtures at the end of the experiment. Values expressed in g/l are the mean ± SE of three replicates. Wastewater
Control at start
Control at the end
Local isolate
S. dimorphus
S. minutum
a b c
0.31 ± 0.01 0.02 ± 0.002 0.02 ± 0
0.35 ± 0.01 0.08 ± 0.01 0.07 ± 0.01
0.64 ± 0.04 0.62 ± 0.01 0.7 ± 0.11
1.24 ± 0.03 0.92 ± 0.12 0.86 ± 0.18
1.49 ± 0.09 0.83 ± 0.09 1.12 ± 0.02
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Table 2 Nutrients, total COD, supernatant COD and pH at the beginning of the experiment in the wastewater mixtures. Values expressed in mg/l are the mean ± SE of two replicates. WW mixture
NO3-N mg/l
NH4-N mg/l
Tot N mg/l
PO4-P mg/l
Tot P mg/l
COD tot
COD S
pH
A B c
1.06 ± 0.01 1.08 ± 0.05 1.25 ± 0.06
22.35 ± 0.25 14.75 ± 0.05 21 ± 0.4
44.65 ± 2.05 22.65 ± 2.15 25.1 ± 0.3
10.1 ± 0.1 1.6 ± 0.005 2.99 ± 0.02
12.75 ± 0.05 1.74 ± 0.08 3.18 ± 0.00
1905 ± 25 1325 ± 0 1167 ± 22
278 ± 1 167 ± 3 224 ± 2.5
7.37 7.48 7.73
Table 3 Lipids extracted as % of sample dry weight of three algal strains grown on three different wastewater mixtures. Values are the mean ± SE of three replicates. Wastewater
Local isolate
S. dimorphus
S. minutum
a b c
17.4 ± 0.39 28.1 ± 1.1 25.0 ± 4.3
19.5 ± 1.3 24.0 ± 3.9 25.7 ± 1.0
27.9 ± 0.9 37.2 ± 3.0 32.0 ± 0.3
Fig. 3. Nutrient removal by the three strains used expressed as a percentage of the starting concentration in wastewater mixture (c). Bars represent the mean ± SE of three replicates.
Table 4 Nitrogen concentration as % of the harvested biomass dry weight of the three strains in the three different wastewater mixtures. a + C stands for algae biomass nitrogen concentration plus control biomass; while a C stands only algae biomass given by the final biomass minus the control biomass. NA stands for not available.
Fig. 1. Nutrient removal by the three strains used expressed as a percentage of the starting concentration in wastewater mixture (a). Bars represent the mean ± SE of three replicates.
Fig. 2. Nutrient removal by the three strains used expressed as a percentage of the starting concentration in wastewater mixture (b). Bars represent the mean ± SE of three replicates.
Wastewater
Control at the end
Local isolate
S. dimorphus
S. minutum
a+C aC b c
7.68 ± 0.10 – NA NA
5.92 ± 0.14 4.81 ± 0.26 3.63 ± 0.05 3.84 ± 0.67
3.50 ± 0.28 2.32 ± 0.27 2.52 ± 0.55 3.35 ± 0.59
3.05 ± 0.06 1.97 ± 0.09 2.62 ± 0.34 2.49 ± 0.08
(Golueke et al., 1957; Zhong et al., 2012). In general the removal trends for ammonium, total nitrogen, phosphate and total phosphorus were similar for all the strains in all the wastewater mixtures used (Figs. 1–3). Major variations were apparent in nitrate removal (Figs. 1–3); however, nitrate was present in such low concentrations that a small change will greatly influence the overall trend. The nitrogen concentration of the harvested biomass from wastewaters (b) and (c) confirmed that ammonium had been taken up and not stripped into the atmosphere through flue gas bubbling (compare Tables 1, 4 and 2). In the case of wastewater (a), even though the ammonium and total nitrogen present in the supernatant were drastically reduced (Fig. 1), it can be speculated that a portion of the nitrogen contained in the original biomass was released into the solution over time and, consequently, some of the nitrogen was probably stripped away by bubbling the flue gases. In another study it was found that algae growing in municipal effluent bubbled with flue gases had a biomass yield approximately three times higher than algae bubbled with air; the latter had a very large proportion of ammonia stripped into the atmosphere (unpublished results). Ammonia stripping under air bubbling in small bottles has been shown in a previous study (Talbot and de la Noüe, 1993).
F.G. Gentili / Bioresource Technology 169 (2014) 27–32
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2001; Slade et al., 2004). Furthermore, the constantly increasing interest in growing algae will lead to an increased demand for water and nutrients. Hence the present study showed that pulp and paper wastewater mixed with dairy sludge/effluent or municipal wastewater can be used and treated by algae without the addition of artificial fertilizers. 4. Conclusions
Fig. 4. Total COD removal by the three strains used expressed as a percentage of the starting concentration in three wastewater mixtures. Bars represent the mean ± SE of two replicates.
The interest in growing algae as sources of biomass and bioenergy is constantly increasing. The cultivation of algae demands large quantities of water and nutrients. The use of wastewater to grow algae is particularly interesting from the point of view of sustainability. However, not all types of wastewater are suitable for algae cultivation. This study shows how mixing different types of wastewater, both municipal and industrial, makes it possible to produce algae biomass with a high lipid content while at the same time treating the wastewater with added flue gases. Acknowledgements
The pH for all treatments at final harvest ranged from 7.71 to 8.39. Total COD was greatly reduced in wastewater mixture (c) with S. minutum, and in wastewater mixture (a) with the local strain from 67% to 92.7% respectively (Fig. 4). However this great reduction of the total COD is due to the harvesting by centrifugation that remove both the algae and the particles present in the wastewater mixtures. Considering COD in the supernatant, the highest reduction of 49.8% was reached in wastewater mixture (a) with the local strain (Fig. 5). In wastewater mixture (c) all algae had a negative effect on COD of the supernatant (Fig. 5). The algal strain S. minutum had a negative effect on supernatant COD in all three wastewater mixtures (Fig. 5). A negative impact of algal growth on COD has been previously reported in different municipal wastewaters and swine manure (Wang et al., 2010; Min et al., 2014). Wang et al. (2010) suggested that the algae excreted organic compounds in the growing substrate. Considering that in the present study the algae grew in autotrophic regime with continuous flue gases bubbling and light irradiation then organic molecules have been excreted into the liquid culture. In conventional wastewater treatment processes of the pulp and paper industry, nutrients have been added to ensure microorganisms growth for treating the wastewater (Thompson et al.,
Fig. 5. Supernatant COD removal by the three strains used expressed as a percentage of the starting concentration in three wastewater mixtures. Bars represent the mean ± SE of two replicates.
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