Bioresource Technology 154 (2014) 297–304

Contents lists available at ScienceDirect

Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Biodiesel production from indigenous microalgae grown in wastewater Oladapo Komolafe a, Sharon B. Velasquez Orta b, Ignacio Monje-Ramirez c, Isaura Yáñez Noguez c, Adam P. Harvey b, María T. Orta Ledesma c,⇑ a

School of Civil Engineering and Geosciences, Newcastle University, Newcastle upon Tyne NE1 7RU, England, UK School of Chemical Engineering and Advanced Materials, Newcastle University, Newcastle upon Tyne NE1 7RU, England, UK c Instituto de Ingeniería, Coordinación de Ingeniería Ambiental, Universidad Nacional Autónoma de México, Apartado Postal 70-472, Coyoacán 04510, DF, Mexico b

h i g h l i g h t s  Desmodesmus sp. and mixed culture of microalgae were cultivated in untreated wastewater.  We report effective nutrients and total coliform removal from wastewater by microalgal treatment.  Ozone-flotation was investigated for microalgae harvesting and microalgae cell wall treatment.  The lipid content and FAME conversion of different microalgae strains were compared.  The FAME obtained from ozone-treated microalgae biomass showed more oxidative stability.

a r t i c l e

i n f o

Article history: Received 27 September 2013 Received in revised form 6 December 2013 Accepted 11 December 2013 Available online 18 December 2013 Keywords: Biodiesel Microalgae Desmodesmus sp. Wastewater Ozone-flotation

a b s t r a c t This paper describes a process for producing biodiesel sustainably from microalgae grown in wastewater, whilst significantly reducing the wastewater’s nutrients and total coliform. Furthermore, ozone-flotation harvesting of the resultant biomass was investigated, shown to be viable, and resulted in FAMEs of greater oxidation stability. Desmodesmus sp. and two mixed cultures were successfully grown on wastewater. Desmodesmus sp. grew rapidly, to a higher maximum biomass concentration of 0.58 g/L. A native mixed culture dominated by Oscillatoria and Arthrospira, reached 0.45 g/L and exhibited the highest lipid and FAME yield. The FAME obtained from ozone-flotation exhibited the greatest oxidative stability, as the degree of saturation was high. In principle ozone could therefore be used as a combined method of harvesting and reducing FAME unsaturation. During microalgae treatment, the total nitrogen in wastewater was reduced by 55.4–83.9%. More importantly, total coliform removal was as high as 99.8%. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Microalgal biodiesel has been reported to have the most potential to displace petroleum derived fuels without negatively affecting food and crop production, since crop-derived biodiesel and bioethanol are deemed unsustainable (Singh and Dhar, 2011). Biodiesel production from microalgae biomass has been suggested to be promising as microalgae have improved characteristics over crops and plant sources, such as: increased photosynthetic efficiency, shorter reproduction cycle, higher nutrients absorption efficiency, lipid content, and biomass productivity (Jaimes-Duarte et al., 2012; Mata et al., 2010). Additional microalgae advantages over land crops or plants for biodiesel production include: microalgae can be cultivated in ⇑ Corresponding author. Tel.: +52 5556233600x3672; fax: +52 5556162164. E-mail address: [email protected] (M.T. Orta Ledesma). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.12.048

wastewater or seawater thereby reducing clean water requirements and the carbon footprints associated with water treatment (Rodolfi et al., 2009), microalgae can obtain nutrients when cultured in wastewater which eliminates fertilizer and herbicide requirements and the pollution effects associated with them (Mata et al., 2010), microalgae can recycle CO2 (Rodolfi et al., 2009), and the residual algae biomass after oil extraction can be used a fertilizer or fermented to produce alcohol (Mata et al., 2010). Unfortunately, microalgal biodiesel production at a commercial scale is not yet feasible because of its high cost, the efficiency of the process must be improved for microalgal biodiesel to compete or displace petro-diesel (Williams and Laurens, 2010; Chisti, 2007). Williams and Laurens (2010) carried out a cost analysis on microalgae biodiesel production and concluded that, with the price of biodiesel at $200 per barrel, the process can only be profitable if other biochemical products contained in the microalgae biomass are processed and marketed; especially marketing protein as an

298

O. Komolafe et al. / Bioresource Technology 154 (2014) 297–304

animal feed. Additionally, marketing some high value by-products from biofuel generation using microalgae biomass can make this technology more economically feasible; by-products such as eicosapentaenoic acid (EPA) sells for $2,194 per kg (99% purity) and $185 per kg (50–70% purity) (Alabi et al., 2009). Growing algae in wastewater offers numerous economic and environmental merits, providing one of the most sustainable ways to produce biodiesel derived from microalgae. Wastewater usage eliminates competition for fresh water, saves cost of nutrients supplement since nutrients are in abundance in wastewater, provides the treatment of the wastewater by assimilating organic and inorganic pollutants into their cells, and eliminates the CO2 emissions associated with wastewater treatment (Chinnasamy et al., 2010; Samori et al., 2013; Wang et al., 2010). The current price of agricultural fertilizers like urea, di-ammonium phosphate and potash are about $420, $480 and $400 per tonne, respectively (Bain, 2013). Hence, substantial savings can be made on nutrients requirement when wastewater is utilised for culturing microalgae. Biodiesel production coupled with wastewater treatment is a promising solution to reduce the economic and environmental cost of biodiesel production (Zhou et al., 2012). The U.S annually invests $13–$21 billion on wastewater infrastructure and $21.4–$25.2 billion for operation and maintenance according to the Government of US (2000). Microalgal wastewater treatment is based on the assimilation of the pollutants into cell constituents like lipids and carbohydrates (Wang et al., 2010). Microalgae can grow well in municipal, domestic and agricultural wastewater. A review on microalgae wastewater treatment by Abdel-Raouf et al. (2012) stated that microalgae can reduce BOD, remove nutrients (nitrogen and phosphorus), coliforms and heavy metals from wastewater. Chinnasamy et al. (2010) cultured a mixed microalgae consortium in carpet mill wastewater and achieved 6.82% oil yield and 96% nutrient removal. Wang et al. (2010) used chlorella for wastewater treatment and obtained high nitrogen, phosphorus, and chemical oxygen demand (COD) removal within 10 h – 42 days of retention time. Ruiz-Martinez et al. (2012) obtained 67.2% and 97.8% ammonia and phosphate removal from the effluent of anaerobic membrane bioreactor by culturing mixed cultures of microalgae in the effluent. Wu et al. (2012) also reported that cultivation of Chlamydomonas sp. in raw industrial wastewater removed 100% ammonia and nitrate, and 33% of phosphate. Recently, Sacristan de Alva et al. (2013) cultured the microalgae Scenedesmus acutus in pre- and post-treated municipal wastewater. The highest level of nutrient removal was found in the pre-treated wastewater (66% of phosphorus and 94% of organic nitrogen). Likewise, better results on biomass productivity and lipid accumulation were found in cultures using pre-treated wastewater. In order to increase the feasibility of biodiesel from microalgae, apart from using fast growing and high lipid producing microalgae strains (Chisti, 2007), the availability of the lipids should be carefully considered. Sometimes the microalgae structure does not allow to extract all lipids in the transesterification process for the production of fatty acid methyl esters (FAME). One way to increase the amount of FAME produced is to use unit operations that make the microalgae lipids easily available. In this instance, ozoneflotation is a harvesting technology that helps increase the lipid availability (Nguyen et al., 2013) and it may be to a point where process costs are reduced. Evaluating and optimising ozoneflotation could offer an alternative to increase the commercial viability of microalgae fuels as it has been desired in previous literature (Chen et al., 2011b; Pragya et al., 2013). The effect of ozone-separated algae biomass on the lipid content, FAME yield and composition, has not been previously reported in literature. Cheng et al. (2011) and Nguyen et al. (2013) all focused on the effects of ozone on microalgae harvesting and recommended future

research on assessing the effects of ozone pre-oxidation in the lipid content and its extraction from microalgae cells. Therefore, this paper provides insight on the effect of ozone application on the lipid content, FAME yield and composition. The aim of this work was to study the production of biodiesel from wastewater grown microalgae by investigating microalgae cultivation and the effect of ozoneflotation over lipid availability and transesterification conversion. 2. Methods 2.1. Wastewater and microalgae characterisation All analyses were carried out in duplicate. Physicochemical and biological properties of the wastewater used as the growth media for microalgae culturing were analysed. Wastewater was characterised according to the parameters shown in Table 1 using standard methods (APHA-AWA-WEF, 2005), phosphorous was expressed as ortophosphates as this is the preferred form assimilated by algae. The microalgae used were native of the ‘‘Lago Nabor Carrillo’’ (an artificial lake) located in Texcoco, Mexico (coordinates 19.4194oN, 99.1333oW). The ‘‘Lago Nabor Carrillo’’ is fed by effluent of the facultative lagoon wastewater treatment plant (FLWTP) also located in Texcoco, Mexico. On May 2013, 100 L of lake water were sampled and collected in several 20 L containers with screw caps and immediately stored at 4 °C. The growth medium (defoamed raw wastewater influent) was collected at FLWTP on the same date. The raw wastewater influent was used as the only growth medium because of the sufficient presence of essential nutrients required for microalgae growth (nitrogen and phosphorus, Table 1). The genera of microalgae present in mixed cultures reactors were investigated under a microscope (Leitzlaborlux S, Germany) using 10, 20, 40 and 100 objectives. Images were taken with a digital camera attached to the eyepiece. 2.2. Experimental set-up Three 20 L open batch reactors were set up for culturing microalgae; open batch reactors were selected to simulate the open pond cultivation method. The open pond system was selected as it is easier to operate and require less capital than photo-bioreactors (Chisti, 2007). A pump (Art: 5060, 5W, Ecopet) was used to aid mixing and to have an adequate distribution of nutrients in the reactors. The reactors were open to atmospheric sunlight and air, and a fluorescent light (MAGGMR 80W tube) was installed to ensure that light supply was not a limitation, the fluorescent light was turned on at day and off at night (12 h on, 12 h off). Reactor 1 and Reactor 3 were used to cultivate the mixed culture of microalgae taken from the lake water. Reactor 2 was used to cultivate Desmodesmus sp. isolated from the lake water. The initial conditions were not the same for all reactors because the algae samples utilised were abstracted from a natural lake, some of which were isolated and sustained in wastewater. Hence the algae already contained some nutrients, this was considered in the assays to have the real total nutrient (nitrogen and phosphate) content in the system at the beginning of the cultivation period, and to compare it with the concentration levels after cultivation and microalgae separation according to the standard method. During the experiment Reactor 1 was found to be dominated by cyanobacteria (Oscillatoria and Arthrospira) while Reactor 3 was found to be dominated by Desmodesmus sp. 2.3. Determination of algal growth Biomass concentration (as total suspended solids, TSS) and optical density (as absorbance) were used to monitor microalgae

O. Komolafe et al. / Bioresource Technology 154 (2014) 297–304 Table 1 Characteristics of the raw wastewater used as the growth media.

a

Parameter

Concentration in wastewater

Strengtha

Total suspended solids (TSS) (mg/L) Volatile suspended solids (VSS) (mg/L) % VSS in TSS Biochemical oxygen demand (BOD5) (mg/O2L) Total coliforms (CFU/100 ml) Total Kjehdal nitrogen (TKN) (mg N/L) Ammonia nitrogen (mg N/L) Organic nitrogen (mg N/L) Nitrate (mg N/L) Nitrite (mg N/L) Phosphorus as orthophosphates (mg PO43-P/L)

160 ± 4.7 146.7 ± 0.007 91.7 ± 2.7 108.3 ± 6.3

Weak Weak – Weak

3  107 ± 1.4  106 42.3 ± 0.4 29.12 ± 0.0 13.2 ± 0.4 23.1 ± 0.6 0.16 ± 0.02 35.4 ± 0.1

Medium – Medium Medium – – High

Classified according to Metcalf and Eddy (1991).

growth. Absorbance was measured at 680 nm using a HACH 3900 spectrophotometer and correlated to TSS. The change in absorbance was calculated by subtracting the initial wastewater absorbance from the absorbance obtained at certain time. Measurements were performed in duplicate. Microalgae productivity rate (g/Ld) in each reactor was estimated through the biomass concentration variation between the initial time (day 0) and the time of highest biomass concentration according to the equation below:

P¼

ðX 1  X o Þ t1  t0

where X1 represents the biomass concentration at the peak concentration (g/L), X0 represents the initial biomass concentration (g/L), t1 represents the day the peak biomass concentration was achieved, and t0 is the day initial biomass concentration was obtained. Cell counting was also used to monitor microalgae growth according to the APHA standard methods (APHA-AWA-WEF, 2005). The results were displayed in cells/ml. 2.4. Biomass harvesting and treatment ‘‘The choice of preferable harvesting technology depends on the algae specie, size, density and desired end product’’ (Pragya et al., 2013). Ozone separation also depends on the biomass concentration, cell size, the buoyancy and cell wall rigidity of the microalgae. For this reason the ozone conditions were different for microalgae strains investigated. The applied ozone dose for reactor 1 was 150 mgO3/L of water, 300 mgO3/L + 5 min of aeration for reactor 2, and 300 mgO3/L for reactor 3. Ozone separation was followed by centrifugation (10,733g for 5 min). Ozone-flotation with centrifugation harvesting process was termed process flow 1. The remaining biomass was separated by sedimentation (48 h) and centrifugation (22,056g for 30 min) to enable comparison with the ozone-harvested sample. The sedimentation with centrifugation harvesting process was termed process flow 2. 2.5. Analyses 2.5.1. Lipid extraction Lipid was extracted from the harvested microalgae biomass in process flow 1 (ozone-flotation with centrifugation) and process flow 2 (sedimentation with centrifugation) using a modified Bligh & Dyer method (Bligh and Dyer, 1959). This method was modified by washing the crude lipid extracts with 0.88% KCl solution rather than with water, to minimise loss of lipids during washing (Garces and Mancha, 1993).

299

2.5.2. In-situ transesterification In-situ transesterification was carried out on the microalgae biomass using a version of the Garces and Mancha’s method (1993). Garces and Mancha’s method was modified significantly to improve process efficiency. A methylating mixture containing methanol, toluene, 2,2-dimethylpropane and sulphuric acid using a volume ratio of 39:20:5:2 by volume. 3.3 ml of the methylating mixture was mixed with 1.7 ml of heptane and added to 0.2 g of dried microalgae biomass in a glass tube. The tube was placed in an incubator (IKA KS 4000i) for 24 h, at 60 °C, and rotation rate of 200 rpm for intense mixing. After the reaction, the sample was cooled down to room temperature and the two layers were allowed to separate. The upper layer containing the FAME was extracted using a Pasteur pipette into a weighted Eppendorf tube. The filled tube was weighed and the mass subtracted from that of the empty tube to obtain the weight of the product. Reactions were carried out in duplicate per reactor containing different microalgae strains. 2.5.3. FAME profiling Gas chromatography (GC Hewlett Packard 5890 series II) was used to profile the FAME produced. 250 mg of sample were mixed with 100 ll of the standard solution (C17:0 Sigma–Aldrich, 10 mg/ml) in a 2 ml vial. 3 ll of methanol were injected into the GC for cleaning, followed by 3 ll of standard solution (C17:0) to confirm standard legibility. After this, 3 ll of the sample and standard mixture were injected into the GC followed by 3 ll of the grain FAME mix (Sigma–Aldrich, 10 mg/ml) for the purpose of peak identification. Finally, 3 ll of methanol was injected into GC for cleaning. Helium (carrier gas), air and hydrogen input pressures were set at 7, 32 and 22 psi, respectively and column head pressure was fixed at 4.5 psi. The run time was for about 30 min. 2.5.4. Statistical analysis MinitabÒ, version 16 statistical software was used to perform a variance analysis using one way ANOVA. The method was used to determine if there was a significant difference in absorbance among reactors containing different types of microalgae strains. The absorbance data obtained was used to monitor microalgae growth in the open batch reactors using untreated wastewater. One way ANOVA was also used to compare the overall FAME conversion obtained for the three reactors according to differences in microalgae genera present in the reactors. 3. Results and discussion 3.1. Wastewater characterisation Wastewater characterisation is important to determine nutrient supplements for microalgae growth, and to investigate wastewater treatment by microalgae during the culturing period. Table 1 shows the results obtained for the assessed parameters. The concentration of ammonia nitrogen and orthophosphates was 29.12 and 35.4 mg/L, respectively; this concentration differed from that used in some research papers. For example, Wang et al. (2010) used untreated wastewater that contained 33.4 mg/L ammonia nitrogen and 5.66 mg/L of orthophosphates to cultivate Chlorella sp. Zhou et al. (2012) cultivated Auxenochlorella protothecoides in concentrated municipal wastewater containing 134 mg/L of total nitrogen and 211 mg/L of orthophosphates. Hence, wastewater with different nutrient concentrations can be used to grow microalgae. The wastewater utilised also contained a high concentration of total coliforms (3  107 CFU/ml). Unfiltered wastewater was applied directly to cultivate algae instead of filtering the wastewater first as commonly done in previous research work (Wang et al.,

300

O. Komolafe et al. / Bioresource Technology 154 (2014) 297–304

2010; Chen et al., 2011a). This was done to investigate wastewater treatment by microalgae in terms of total coliform removal; since filtration and other pre-treatments remove some bacteria. This also provides ‘more real terms’ data as the large scale production of microalgae in open ponds is likely to be prone to contamination by bacteria and/or fungi. 3.2. Microalgae genera present in the mixed culture reactors Eleven microalgae strains were found in the mixed cultures of reactor 1. Cyanobacteria were the most dominant (i.e., Oscillatoria) genera followed by Arthrospira. In mixed cultures reactor 3, about 6 strains were found and the most dominant genus was Desmodesmus sp., followed by Navicula. Other genuses found in reactor 3 included Oscillatoria, Chlorella, Microcystis and Chlamydomonas. Cyanobacteria cells are coccoid or rod shaped with a length range of 0.5–30 lm, their cell wall is composed of polysaccharides (Garofalo, 2009). Cell numbers of cyanobacteria have been found to double within 24 h, but some Oscillatoria strains double in 4 h (Castenholz and Waterbury, 1989). Oscillatoria and Arthrospira are genera in the phylum cyanobacteria, and they are fast growing. It has been reported that Arthrospira contains 55–77% dry weight of protein (Tokusoglu and Unal, 2003), and is rich in poly unsaturated fatty acids such like c-linolenic acid (Garofalo, 2009). Desmodesmus sp. are medium to large size green algae with an ellipsoidal or ovoid cell shape. Under exponential growing conditions, the cells contain about 50% protein, 10% lipids, starch, and cell wall components. However, under stress conditions, the lipid content is increased (to about 30%) and accumulates mostly C16 and C18 unsaturated and some monounsaturated fatty acids (Garofalo, 2009), C16 fatty acid is suitable for biodiesel production. Desmodesmus sp. are among the fastest growing microalgae strains in wastewater and produce high oil yields (Rodolfi et al., 2009; Hu et al., 2008). According to literature, diatoms are also promising for biodiesel production. Diatoms produce majorly C16 and C16:1 fatty acids and highly nutritional biomolecules like poly unsaturated fatty acids (Hu et al., 2008). Mixed culture of microalgae were selected as one of the strains to be investigated because several microalgae genera are often found together; therefore, the cost and energy to isolate and purify individual microalgae species can be saved, and there would be no worries of strain contamination during culturing. Desmodesmus sp. was solely inoculated in reactor 2 because it has been reported as a high growing genus in wastewater by Rodolfi et al. (2009) and Hu et al. (2008). 3.3. Microalgae growth All microalgae strains investigated were able to absorb nutrients, from the wastewater, for growth. For a commercial process, nutrient supplement costs could therefore be reduced or eliminated since no additional nutrients were added to the cultivation media. The monitoring of growth using absorbance and TSS showed the same trend and a correlation (R2) of 0.75, 0.77 and 0.97 for reactor 1, 2 and 3, respectively. Although the correlation between TSS and absorbance for reactor 1 and reactor 2 is low; both the absorbance and TSS curves indicated the same maximum steady growth. Cell residence time was determined by the maximum steady state point achieved in the microalgae growth curve. The cell residence time for reactors, containing varied microalgae strains, was not the same because of differences in nutrient consumption and cell duplication. The cell residence time for reactor 1 (cyanobacteria dominated mixed culture of microalgae), reactor 2 (Desmodesmus sp.) and reactor 3 (Desmodesmus sp. dominated mixed culture of microalgae) microalgae strains were 8, 5 and 19 days, respectively (Table 2). Desmodesmus sp. solely cultivated

in reactor 2 grew faster than the other microalgae strains achieving the highest maximum biomass concentration of 0.58 g/L in the shortest residence time as shown in Table 2. This indicates that, in terms of biomass production, culturing Desmodesmus sp. from the beginning was more efficient than initially seeding mixed cultures. The results support findings from Rodolfi et al. (2009) and Hu et al. (2008); that Desmodesmus sp. are one of the fastest growing microalgae in wastewater. Therefore, Desmodesmus sp. have the highest biodiesel production potential in terms of biomass yield. More biomass means biochemical products like bioethanol and proteins can be produced in substantial quantities and marketed. This supports the cost analysis report presented by Williams and Laurens (2010) that extracting and marketing other useful biochemical products is essential to a sustainable biodiesel production. Ammonia was the preferred nitrogen source by all the microalgae strains as complete depletion was experienced after a few days while phosphate was utilised in moderate quantities. The pH and dissolved oxygen concentration increased as the microalgae grew in the three reactors; this further indicated that the microalgae strains were indeed growing in the wastewater medium. The growth rate of reactor 1 mixed culture of microalgae (cyanobacteria dominated), reactor 2 (Desmosdesmus sp.) and reactor 3 mixed culture of microalgae (Desmodesmus sp. dominated) were compared to determine the difference or similarity on how they grew under the subjected conditions in relation to each other. Fig. 1 demonstrates the changes in microalgae growth for reactors. The boxes indicate the interquartile range of the data (the upper box represents the 25th percentile and the lower box represents the 75th percentile), the mean is represented by the middle line bisecting the two boxes, the minimum value is represented by the bottom of the error bar and the tip of the error bar on the boxes represents the maximum value. One-way ANOVA, used to determine the difference in absorbance among reactors, produced a p-value of 0.00 which implies that the change in absorbance for each reactor varied. This difference might be due to the variation in the absorption of nutrient and ability to grow in wastewater by the microalgae strains. 3.4. Wastewater treatment by microalgae Microalgae has the ability to remove nutrients (nitrogen and phosphorus), and hence reduce chemical oxygen demand (COD) from wastewater. Treatment of wastewater with algae resulted in better nutrient removal than the conventional activated sludge system. Algae treatment is applied in some treatment works, after an activated sludge system, as a tertiary treatment used to comply with discharge standards (Tam and Wong, 1989). It may be beneficial to completely replace the activated sludge system with algae based treatment system for simultaneous nutrients and COD removal rather than only being used as a tertiary treatment. Wastewater treatment by microalgae in the three reactors was assessed based on total nitrogen, total coliform and orthophosphates removal. Total nitrogen is the sum of the TKN, nitrate, and nitrite concentration. The initial concentration of these parameters were measured in the wastewater before algae inoculation and after algae cultivation period to determine the treatment achieved in each reactor. Fig. 2a shows that algae cultivation in wastewater was effective in reducing the total nitrogen concentration in wastewater. Mixed culture microalgae in reactor 3 (Desmodesmus sp. dominated) reduced the total nitrogen in wastewater by 83.9% over 22 days followed by Desmodesmus sp. in reactor 2 with percentage removal of 80% in 22 days. Mixed culture of microalgae (cyanobacteria dominated–Oscillatoria and Arthrospira) in reactor 1 reduced the total nitrogen by 55.4% but in a shorter residence time of 11 days. This

301

O. Komolafe et al. / Bioresource Technology 154 (2014) 297–304 Table 2 Summary of the growth parameters for the microalgae strains investigated.

a

Parameter

Mixed cultures microalgae-cyanobacteria dominated (Reactor 1)

Desmodesmus microalgae (Reactor 2)

Mixed cultures microalgae-Desmodesmus dominated (Reactor 3)

Max. biomass concentration (g/L) Biomass productivity (g/L.d) Cells residence time (days)a pH range Dissolved oxygen range (mg/L)

0.36 ± 0.02 0.017 8 8.7–10.7 4.6–9.5

0.58 ± 0.04 0.013 5 9.5–10.5 6.9–13.0

0.45 ± 0.03 0.017 19 9.3–10.5 5.3–14.2

Is the time at which the microalgae strains grew rapidly before the stationary or death phase sets in the growth curve.

means that reactor 1 has a higher removal rate during the short microalgal growth obtained. The type of microalgae to be selected, in terms of wastewater treatment, will depend on the removal rate required and hydraulic residence time permitted. The orthophosphate concentrations also decreased during cultivation of algae, although to a lesser extent than nitrogen, as shown in Fig. 2b. This was attributed to the minimum nutritional requirements of phosphorus by microalgae, which can be derived from the molecular formula of microalgal biomass, CO0.48H1.83N0.11P0.01 (Chisti, 2007). The mixed culture of microalgae (Desmodesmus dominated) in reactor 3 achieved the highest orthophosphates removal of 61% in 22 days, followed by Desmodesmus sp. in reactor 2 that removed 38% orthophosphates from the wastewater in 16 days, and the mixed microalgae cultures (cyanobacteria dominated) in reactor 1 that achieved 30.1% reduction in 11 days. Total coliform removal from wastewater was effective during algae cultivation as shown in Fig. 2c. Complete removal was achieved by mixed cultures in reactor 1, 99.8% (2-log removal) by Desmodesmus in reactor 2, and 90% (1-log removal) by mixed cultures of microalgae in reactor 3. The total nitrogen concentration of the effluent treated by algae was 7.5 mg/L for reactor 2 and 9.3 mg/L for reactor 3; showing a superior treatment efficiency than conventional treatment. Usually, the effluent from conventional activated sludge treatment system has a total nitrogen concentration between 15 and 35 mg/L which most times does not comply with discharge standards (Carey and Migliaccio, 2009). The nitrogen concentration for reactor 1 effluent (16.8 mg/L) was not superior that in conventional treatment. The acceptance of effluent quality from this algal treatment system is solely based on the intended usage of the effluent. Potential uses include; agriculture use for restricted and unrestricted irrigation or discharging into a water body. For the purpose of discharging into water body in Europe for example, the EU 91/271/EEC Directive sets the total nitrogen limit concentration at 15 mg/L, this concentration was met by algal treatment in reactor 2 (7.5 mg/L) and reactor 3 (9.3 mg/L). However, orthophosphates removal was generally poor, which can be attributed to the release of assimilated phosphorus back into the medium during microalgae death (Uehlinger, 1986); indicating the importance of controlling cell residence time. An appropriate residence time will enable separation of the algae from the wastewater before algae cell death, improve phosphorus removal and gain the highest biomass concentrations for biodiesel (value added fuel) production. The complete total coliform removal in reactor 1, indicates complete faecal coliform removal. Some of the total coliform contained in the effluents from reactor 2 and 3 may have a concentration of faecal coliform above allowable limits; hence faecal coliform an assay is recommended to ascertain its usability. Total coliform removal in wastewater is also very important when the effluent is discharged into bathing waters.

Fig. 1. Box plot of change in absorbance for the reactors (the negative change in absorbance observed was due to the lag phases in the growth curves).

3.5. Lipid content of the microalgae biomass Microalgae strains with high lipid contents are essential for feasible biodiesel production. A significant number of algae strains

Fig. 2. Total nitrogen, orthophosphate and total coliform before and after microalgae treatment. Initial measurements were done after inoculation.

302

O. Komolafe et al. / Bioresource Technology 154 (2014) 297–304

Table 3 Fatty acid profile for the microalgae cultures from the reactors with different harvesting method using GC–FID (In-situ transesterification). FAME type

C8:0 Caprilic C10:0 Capric C12:0 Lauric C13:0 Tridecanoic C14:0 Myristic C14:1n9c Myristoleic C15:0 Pentadecanoic C16:0 Palmitic C16:1n9c Palmitoleic C18:0 Stearic C18:1n9t Oleic C18:2n6c Linoleic C18:3n3 Linolenic C20:0 Arachidic C20:1 Gadoleic Saturated* Mono-unsaturated* Poly-unsaturated* C16–C18*

mg of FAME produced Reactor 1 (PF1 biomass)

Reactor 1 (PF2 biomass)

Reactor 2 (PF1 biomass)

Reactor 2 (PF2 biomass)

Reactor 3 (PF1 biomass)

Reactor 3 (PF2 biomass)

0.03

0.02

0.02

0.09

0.06

0.04

0.13

0.22

0.02

0.11

0.03

0.03

0.05

–

0.02

–

0.06

0.03

0.04

0.09

0.05

0.032

0.17

0.07

0.17

0.16

0.12

–

0.27

0.14

0.20

0.14

0.20

0.35

0.25

0.25

0.07

0.05

0.08

–

0.08

0.05

2.19

2.24

1.07

0.42

0.92

0.79

0.64

0.65

0.15

0.40

0.36

0.47

0.21

0.15

0.07

0.69

0.16

0.07

0.49

0.20

0.59

0.42

0.30

0.24

0.31

0.85

0.49

0.27

0.43

0.50

0.30

1.22

0.06

0.58

0.05

0.09

0.09

–

0.03

–

–

0.25

0.12

0.09

0.07

0.18

0.21

0.12

59.24% 28.73% 12.08% 82.2%

48.51% 17.99% 34.25% 88.05%

48.76% 33.28% 18.12% 99.12

37.96% 38.19% 34.05% 78.64%

51.85% 33.19% 14.22% 65.78%

46.96% 34.50% 18.85% 69.01%

*

Calculation based on the percentage of the interested FAME (mg) in the total FAME (mg); PF 1 and PF 2 means process flow 1 and process flow 2 respectively. Reactor 1 (cyanobacteria dominated mixed cultures of microalgae), reactor 2 (Desmodesmus sp.) and reactor 3 (Desmodesmus dominated mixed cultures of microalgae).

Fig. 3. Comparing the lipid content of biomass recovered with or without ozone for all reactors.

have been shown to contain lipid content of about 20–50% by weight of algae biomass (Chisti, 2007). Different algae species vary in their lipid productivity depending on their photosynthetic system and adaptation to the culture media. Mixed culture of microalgae (cyanobacteria dominated) in reactor 1 yielded more lipids per unit mass biomass than reactor 2 and 3 microalgae strains, as it is shown in Fig. 3. This is interesting because cell concentration after culturing microalgae strains in reactor 1 (4  104 cells/ ml) was lower than that for reactor 2 (16  104 cells/ml), and reactor 3 (25  104 cells/ml); one would have expected to obtain more lipids from the reactors with a higher microalgae cell concentration. Ozone-flotation separated biomass (process flow 1) only yielded more lipids in reactor 1 mixed culture of microalgae (cyanobacteria dominated), but process flow 1 and process flow

Fig. 4. Comparison between the FAME conversions of the microalgae strains under two biomass recovery conditions.

2 harvested microalgae biomass yielded similar amount of lipids in reactor 2 (Desmodesmus sp.) and reactor 3 (Desmodesmus sp. and Navicula sp. dominated) as shown in Fig. 3. The results obtained in reactor 1, suggest that the ozoflotation of microalgae, improves lipid extractability. It has been shown that ozone can lyse cells of microalgae (Langlais et al., 1991), which may contribute in step lipid extraction; recent studies by this research group (results to be published) corroborate this ozone effect. In the case of reactor 2 and reactor 3, the ozone dose might have not been sufficient due to the presence of a higher microalgae biomass than for reactor 1 (see Table 2).

O. Komolafe et al. / Bioresource Technology 154 (2014) 297–304

The lipids obtained from both mixed microalgae strains under the two harvesting conditions (Fig. 3) were superior to the 12% lipids obtained by Chinnasamy et al. (2010) from mixed cultures cultivated in untreated wastewater; while the lipids obtained from reactor 2 mixed microalgae cultures were not superior. Mixed cultures grown in reactor 1 produced 26.3% and 20% lipids per weight of dry biomass for process flow 1 and process flow 2, respectively; and the mixed microalgae culture in reactor 3 obtained lipids of 14.29% and 16.08% for process flow 1 and 2, respectively. The lipid contents produced from biomass harvested using process flow 1 and process flow 2 for the Desmosdesmus genus (reactor 2) were 12.9% and 13.3%, respectively (Fig. 3). The lipids obtained were lower compared to 21.2%, 19.7% obtained from Desmosdesmus cultivated in photobioreactors by Jaimes-Duarte et al. (2012) and Wu et al. (2012), respectively. This might be as a result of controlled carbon dioxide, oxygen, light intensity and less interference from the environment in photobioreactors used in previous studies. However, photobioreactors are more expensive than the open batch reactors applied in this work, and can make the culturing process less cost effective. 3.6. In-situ transesterification and GC–FID analysis The amount of FAME obtained when using the Garces and Manchas method is shown in Fig. 4. In general, mixed cultures of microalgae in reactor 1 (cyanobacteria dominated) produced a higher FAME conversion than for the other strains (reactor 2 and 3), under the investigated conditions. However, the FAME conversion of harvested biomass using process flow 1 (ozone-flotation plus centrifugation) was not significantly different among the three reactors (ANOVA p-value of 0.14). Also, there was no significant difference in the FAME conversion (ANOVA p-value = 0.06) when comparing harvested biomass for all reactors using process flow 2 (sedimentation with centrifugation). Palmitic acid (C16:0) was the most abundant FAME in most cases except in reactor 2 (Desmosdesmus sp.) process flow 2 (sedimentation with centrifugation) harvested biomass (Table 3). Palmitic acid (C16:0) was found as the most abundant FAME from Desmodesmus sp. cultured in wastewater by Samori et al. (2013). It was discovered that total saturated FAME was more than total unsaturated FAME (mono and poly unsaturated) for microalgal biomass ozone-separated for all reactors. These results can be explained by the fact that unsaturated lipids (including triglycerides and fatty acids) with two or more double bonds in their structure, favour the reaction with ozone. Ozone oxidizes double-bonded compounds into oxygenated functional groups like ketones and aldehydes (Langlais et al., 1991); therefore, a decrease in the percentage of unsaturated FAME was expected. 3.7. Effect of ozone treatment on FAME composition The effect of ozone-treatment on algae was shown in the degree of saturation or unsaturation of the FAME in the samples. Ozonetreated samples (Process flow 1) have a higher percentage of saturated FAME than the untreated samples (Process flow 2) (Table 3). Higher degree of FAME saturation is desirable from biodiesel produced from microalgae because of its oxidative stability; oxidative stability implies that the FAME will not oxidize to undesirable products like ketonic and carboxylic compounds during storage (Chisti, 2007). Furthermore, the ozone-treated samples obtained a lower degree of poly-unsaturation as shown in Table 3. Untreated samples in all reactors had more poly-unsaturated FAME (higher degree of unsaturation) than the ozone-treated samples. The higher the degree of saturation, the greater the oxidative instability of the FAME; partial catalytic hydrogenation treatment of oil to reduce

303

the extent of unsaturation is recommended for oils with FAME of about 4 double bonds (Chisti, 2007). The FAME produced without ozone treatment had too high a level of linolenates to pass the EU standard. This was significantly reduced by ozone treatment in all cases, which may represent a distinct advantage for this technique. It is suggested that during this oxidation, multiple products are formed; a product with reduced unsaturation (major product) and an oxidized by-product. This might explain how ozone-treated samples produced a higher degree of saturation or a lower degree of unsaturation than the untreated samples. The European Standard EN 14214 sets limits on the amount of some poly unsaturated FAME (like linolenic acid to 12%–mol) for vehicle use (Chisti, 2007), showing the importance of the ozone-treatment on the microalgae strains. 4. Conclusions Wastewater was effectively utilised for culturing microalgae without addition of nutrients. A high degree of nitrogen removal and high total coliform removal (up to 99.8%) were achieved. The native cyanobacteria-dominated mixed cultures yielded more lipids than the pure and Desmodesmus sp. dominated mixed cultures; in addition, the FAME conversion obtained was higher than the other microalgae cultures studied. The FAME produced after ozonation treatment, exhibited a greater degree of saturation. This degree of saturation met the cold filter plugging point (‘‘CFPP’’) standard specification, although here mono- and diunsaturated species remained after ozonation. Acknowledgements Thanks to Miss Maria Teresa Valeriano and Mr Isaac Nava for her support in the ozonation experiments. The help of Miss Andrea Hernandez Garcia in cell counting and algae identification is also greatly appreciated. Mr Oladapo Komolafe appreciates the partial support received through a Tulip scholarship to be able to visit Mexico. This research project was supported by the Institute of Engineering based at UNAM, Mexico through international collaborative funding. References Abdel-Raouf, N., Al-Homaidan, A.A., Ibraheem, I.B.M., 2012. Microalgae and wastewater treatment. Saudi. J. Bio. Sci. 19 (3), 257–275. Alabi, A.O., Tampier, M., Bibeau, E., 2009. Microalgae Technologies & Processes FPR Biofuels/Bioenergy Production in British Columbia: Current Technology, Suitability & Barriers to implementation. British Columbia Innovation Council. APHA-AWA-WEF, 2005. Standard Methods for the Examination of Waters and Wastewater, 21 ed. APHA, Washington, DC. Bain, B., 2013. Fertilizer Outlook. In: Nomura Global Chemical Industry Leaders Conference. FERTECON Limited, Venice. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37 (8), 911–917. Carey, R.O., Migliaccio, K.W., 2009. Contribution of wastewater treatment plant effluents to nutrient dynamics in aquatic systems: a review. Environ. Manage. 44 (2), 205–217. Castenholz, R.W., Waterbury, J.B., 1989. Group 1. Cyanobacteria. In: Staley, J.T., Bryant, M.P., Pfennig, N., Holt, J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology, Vol. 3. Williams & Wilkins, Baltimore. Chen, M., Tang, H., Ma, H., Holland, T.C., Ng, K.Y., Salley, S.O., 2011a. Effect of nutrients on growth and lipid accumulation in the green algae Dunaliella tertiolecta. Bioresour. Technol. 102 (2), 1649–1655. Chen, Chun-Yen, Yeh, Kuei-Ling, Rifka Aisyah, Lee, Duu-Jong, Chang, Jo-Shu, 2011b. Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review. Bioresour. Technol. 102 (2), 71–81. Cheng, Y.L., Juang, Y.C., Liao, G.Y., Tsai, P.W., Ho, S.H., Yeh, K.L., Chen, C.Y., Chang, J.S., Liu, J.C., Chen, W.M., Lee, D.J., 2011. Harvesting of Scenedesmus obliquus FSP-3 using dispersed ozone flotation. Bioresour. Technol. 102 (1), 82–87. Chinnasamy, S., Bhatnagar, A., Hunt, R.W., Das, K.C., 2010. Microalgae cultivation in a wastewater dominated by carpet mill effluents for biofuel applications. Bioresour. Technol. 101 (9), 3097–3105. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306.

304

O. Komolafe et al. / Bioresource Technology 154 (2014) 297–304

Garces, R., Mancha, M., 1993. One-step lipid extraction and fatty acid methyl esters preparation from fresh plant tissues. Anal. Biochem. 211 (1), 139–143. Garofalo, R., 2009. Algae and aquatic biomass for a sustainable production of 2nd generation fuels AquaFUELS-Taxonony. Biol. Biotech. 1–128. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., Darzins, A., 2008. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54 (4), 621–639. Jaimes-Duarte, D., Soler-Mendoza, W., Valasco-Mendoza, J., Munoz-penaloza, Y., Urbina-Suarez, N., 2012. Characterization chlorophtas microalgae with potential in the production of lipids for biofuels. CT&F–Cienc. Technol. Futuro. 5, 93–102. Langlais Bruno, Reckhow David A, Deborah R. Brink, 1991. Ozone in Water Treatment, Application and Engineering. Ed Lewis publishers, USA. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae for biodiesel production and other applications: a review. Renew. Sust. Energy Rev. 14 (1), 217–232. Metcalf & Eddy, Inc., 2003. Wastewater Engineering: Treatment and Reuse, 4th ed. McGraw-Hill, New York. Nguyen, T.L., Lee, D.J., Chang, J.S., Liu, J.C., 2013. Effects of ozone and peroxone on algal separation via dispersed air flotation. Colloids Surf. B. Biointerfaces. 105, 246–250. Pragya, N., Pandey, K.K., Sahoo, P.K., 2013. A review on harvesting, oil extraction and biofuels production technologies from microalgae. Renew. Sust. Energy Rev. 14, 159–171. Rodolfi, L., Chini Zittelli, G., Bassi, N., Padovani, G., Biondi, N., Bonini, G., Tredici, M.R., 2009. Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 102 (1), 100–112. Ruiz-Martinez, A., Martin Garcia, N., Romero, I., Seco, A., Ferrer, J., 2012. Microalgae cultivation in wastewater: nutrient removal from anaerobic membrane bioreactor effluent. Bioresour. Technol. 126, 247–253.

Sacristan de Alva, M., Luna-Pabello, V.M., Cadena, E., Ortíz, E., 2013. Green microalgae Scenedesmus acutus grown on municipal wastewater to couple nutrient removal with lipid accumulation for biodiesel production. Bioresour. Technol. 146, 744–748. Samori, G., Samori, C., Guerrini, F., Pistocchi, R., 2013. Growth and nitrogen removal capacity of Desmodesmus communis and of a natural microalgae consortium in a batch culture system in view of urban wastewater treatment: part I. Water Res. 47 (2), 791–801. Singh, N.K., Dhar, D.W., 2011. Microalgae as second generation biofuel. A review. Agron. Sustain. Dev. 31 (4), 605–629. Tam, N.F., Wong, Y.S., 1989. Wastewater nutrient removal by Chlorella pyrenoidosa and Scenedesmus sp. Environ. Pollut. 58 (1), 19–34. Tokusoglu, O., Unal, M.K., 2003. Biomass nutrient profiles of three microalgae: Spirulina platensis, Chlorella vulgaris, and Isochrisis galbana. J. Food Sci. 68 (4), 1144–1148. Uehlinger, U., 1986. Bacteria and phosphorus regeneration in lakes. An experimental study. Hydrobiologia 135, 197–206. Wang, L., Min, M., Li, Y., Chen, P., Chen, Y., Liu, Y., Wang, Y., Ruan, R., 2010. Cultivation of green algae Chlorella sp. in different wastewaters from municipal wastewater treatment plant. Appl. Biochem. Biotechnol. 162 (4), 1174–1186. Williams, P.J.l.B., Laurens, L.M.L., 2010. Microalgae as biodiesel & biomass feedstocks: review & analysis of the biochemistry, energetics & economics. Energy Env. Sci. 3 (5), 554. Wu, L.F., Chen, P.C., Huang, A.P., Lee, C.M., 2012. The feasibility of biodiesel production by microalgae using industrial wastewater. Bioresour. Technol. 113, 14–18. Zhou, W., Li, Y., Min, M., Hu, B., Zhang, H., Ma, X., Li, L., Cheng, Y., Chen, P., Ruan, R., 2012. Growing wastewater-born microalga Auxenochlorella protothecoides UMN280 on concentrated municipal wastewater for simultaneous nutrient removal and energy feedstock production. Appl. Energy 98, 433–440.