Bioresource Technology 121 (2012) 298–303 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech Using ammonia for algae harvesting and as nutrient in subsequent cultures Fangjian Chen, Zhiyong Liu, Demao Li, Chenfeng Liu, Ping Zheng, Shulin Chen ⇑ Tianjin Key Laboratory for Industrial Biological Systems and Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China h i g h l i g h t s " We present a novel ﬂocculation process using ammonia as coagulant. " The novel process is effective for the tested different algae strains. " Ammonia is converted into ammonium using greenhouse gas during the novel process. " The ammonia added will be reused as fertilizer in the subsequent cultures. " Ammonia does not change the metabolic components without metal ions inputting. a r t i c l e i n f o Article history: Received 7 May 2012 Received in revised form 26 June 2012 Accepted 27 June 2012 Available online 3 July 2012 Keywords: Aqueous ammonia Algae Harvest Reuse a b s t r a c t Microalgae have been considered as a promising feedstock for biofuels and greenhouse gas reduction. A low-cost harvesting technology without secondary contamination for down-stream extraction is a key requirement to make algal biofuel commercially viable. A novel harvesting method using ammonia as a ﬂocculant to make the algal biomass settable was devised and studied. Another major advantage of this approach is that the ammonia added will be reused as fertilizer in the subsequent cultures. The results indicated that ammonia-induced ﬂocculation led to more than 99% removal of algae at 12 h. The OD600 of algae growing in the ammonia-enriched ﬂocculation medium treated with heating and CO2 was 2 times than that of initial after 6 days. These results suggested that this ﬂocculation method was efﬁcient, convenient and allowed the reuse of the ﬂocculated medium, therefore providing an option for economic harvesting and cultivation of microalgae. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction With the continuous depletion of petroleum and the rising CO2 concentration in the atmosphere from the combustion of fossil fuels, increasing attention has been given to the development of the alternative fuels, such as lipid-derive biodiesel (Iglesias-Rodriguez, 2008) and renewable hydrocarbons (Graves et al., 2011). Biodiesel has traditionally been produced with a variety of feedstocks such as soybeans, canola oil, animal fat, palm oil, corn oil, waste cooking oil (Felizardo et al., 2006), jatropha oil (Barnwal and Sharma, 2005). These conventional lipid sources not only are subjected to quantity limitation, but also cause concerns in competition with the need as food for human. Algae have attracted a great interest in recent years because of their various desirable characteristics such as high potential productivity (Amaro et al., 2011; Brennan and Owende, 2010; Chisti, 2007). Additionally, algae can be potentially produced with resources such as salt water or wastewater that are not used for food production. Moreover, biofuel production from large-scale ⇑ Corresponding author. E-mail address: [email protected] (S. Chen). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.06.076 cultivation of microalgae has been widely regarded as one of the most promising alternatives that have potential to deal with the problem of global warming (Benemann and Oswald, 1996; Chisti, 2007). Many technical barriers, however, exist to the commercialization of algal biofuel systems among which concentrating the algal biomass is a major challenge (Wang et al., 2008). It was estimated that the cost of harvesting algal biomass was 20–30% of the total costs of production (Gudin and Thepenier, 1986) due to small cell size (typically in the range of 1–30 lm), low concentration (typically in the range of 0.3–5 g/l), unfavourable density (only slightly greater than media) and negative charge (contributing to their stability in a dispersed state) (Brennan and Owende, 2010). Major techniques available for harvesting microalgae include ﬁltration, centrifugation, gravity sedimentation, ﬂocculation (Benemann and Oswald, 1996; Molina et al., 2003) and electrophoresis (Amaro et al., 2011; Danquah et al., 2009). However, rapid clogging, continuous backwashing and the costs arising from pumping and membrane replacement are the major problems in ﬁltration techniques (Becker, 1994; Molina et al., 2003). Centrifugation requires high energy input, complicated processing and F. Chen et al. / Bioresource Technology 121 (2012) 298–303 299 large capital investment (Benemann and Oswald, 1996; Molina et al., 2003). Gravity settling is suitable only to harvest large-sized microalgal cells, e.g. Spirulina spp.(Xiong et al., 2008). Electrolytic processes with increasing system temperature and cathode fouling lead to high power consumption (Amaro et al., 2011). The harvesting of microalgal cells by ﬂocculation is seen to be a superior method to other aforementioned harvesting methods because of its effectiveness with given cost (Pushparaj et al., 1993). Chemicals called ﬂocculants are usually added to induce ﬂocculation followed by gravity separation. Multivalent metal salts like ferric chloride (FeCl3), aluminium sulphate (Al2(SO4)3) and ferric sulphate (Fe2(SO4)3) (Shelef et al., 1984) and certain cationic polymers such as, chitosan, cationic polyacrlyamides, and cellulose, surfactants, and other man-made ﬁbers (Bilanovic et al., 1988; Oh et al., 2001; Pushparaj et al., 1993) have been tested effective. Although ﬂocculation has proven to be successful for concentrating microalgae, a large amount of ﬂocculant is needed to cause solid–liquid separation of the microalgae. The algal biomass as the end product is contaminated by the added ﬂocculant, thus algae harvesting with chemical ﬂocculant is not suitable for biofuel application due the added cost and residual effects on algal biomass and the culture water. An ideal ﬂocculant for microalgae harvesting must meet the following criteria: (i) resulting in no residual in biomass, (ii) leading to high efﬁcient subsequent settling of aglae, (iii) allowing reusing the algal culture medium as growth supporting nutrients for subsequent algal cultivation, (iv) considering the environmental impact, reducing the greenhouse gas effect. A ﬂocculation process using ammonia as coagulant was developed to meet these criteria. In this process, aqueous ammonia is used to alter the pH of the culture that leads to the ﬂocculation and settling of the algae. Upon the removal of the algal biomass, ﬂue gas containing CO2 is introduced to lower the pH to convert the un-ionized ammonia to its ionic form to reduce its potential toxicity when the ammonia laden water is resued for algae culture (Fig. 1). This paper presents the test results of this process, including ﬂocculation efﬁciency, ammonia conversion using CO2, analysis of cell metabolite and morphology, and reuse of the ﬂocculated culture water. Upon further reﬁnement, this process has a potential to provide a viable option for bio-friendly and economic mass cultivation and harvesting of microalgae to make algal biofuel production more competitive. (fresh water), Nannochlropsis oculata (marine), and native algal species (marine, named HTBS, classiﬁed as Dunaliella). C. sorokiniana was cultivated using a Bold’s Basal Medium without glucose (Bischoff and Bold, 1963), the pH of the medium was adjusted to 6.1 before sterilization. The marine algae were cultivated using a f/2 medium (Guillard and Ryther, 1962) with ﬁltered sea water, the composition brieﬂy was: 1.5 g NaNO3, 0.04 g K2HPO4, 0.006 g ferric ammonium citrate, and trace metal mix A5 in 1 L of distilled water without adjusting pH (pH was 8.0). The strains were incubated in plat bioreactor that contained 15 L of the f/2 medium. The algal culture was continuously bubbling sterilized air with 3% CO2 under continuous illumination at 150 lmol m2 s1. The culture temperature was 25 ± 1 °C and the strain was cultivated for 14 days. 2. Methods 2.3. Scanning electron microscopy (SEM) morphology analysis 2.1. Strains and culture medium Scanning electron microscopy (JEOL-2100F, Japan) was used to observe the morphology of HTBS algae cells, and the ﬂocs after the ﬂocculation experiment. HTBS algae cells were placed on glass slides and dried in air. Instead of ﬁxation as traditionally used to The ammonia-based ﬂocculation process was evaluated with both freshwater and marine algae, including Chlorella sorokiniana 2.2. Experimental design and analysis of ﬂocculation efﬁciency In order to assess the ﬂocculation effect of aqueous ammonia, the experiment of HTBS comparing with HTBS-treated algae with aqueous ammonia was ﬁrst carried out. To further analyze the ﬂocculation effect quantitatively, the aforementioned three strains were tested. The cultures were stirred using magnetic stirrer and added different doses of commercial aqueous ammonia by titrimetry. For HTBS the concentrations were 0.09, 0.36, 1.09, 3.12, 12.90, 38.37 mmol L1, For N. oculata the concentrations were 0.74, 2.22, 4.44, 10.72, 26.62, 57.31, 118.7 mmol L1, and for C. sorokiniana the concentrations were 0.52, 4.95, 9.54, 24.26, 48.14, 113.3, 240.6 mmol L1. After the ammonia addition, the removal efﬁciency of ﬂocculated algal cultures was measured at 0.5 h, 1 h, 3 h, 6 h, 12 h. An aliquot was taken at a height of two-thirds from the bottom, and the pH was measured. The optical density (OD) of the aliquot was measured at 600 nm to evaluate the ﬂocculation efﬁciency of the aqueous ammonia (Kim et al., 2011). The ﬂocculating efﬁciency was calculated using the following equation: Flocculating efficiency ð%Þ ¼ ð1 B=AÞ 100 where, A is the optical density of the algal culture before the ﬂocculation measured at 600 nm and B is the optical density of the sample at 600 nm. The supernatant of culture medium was used to carry out reuse experiment. Fig. 1. New harvesting strategy using aqueous ammonia, ﬁrst algae is ﬂocculated and removed after settling, then the supernatant culture medium containing high concentration of NH3H2O, ammonium, hydroxyl ion react with ﬂue gas (10–15% CO2), in which the CO2 and ammonia are converted into bicarbonate and ammonium ion which can be used as the carbon and nitrogen sources, the remains of hydroxyl ion and NH3H2O are greatly reduced, the pH also can be return to normal levels, ﬁnally the culture medium is simply treated and reused to culture the algae. 300 F. Chen et al. / Bioresource Technology 121 (2012) 298–303 Fig. 2. Flocculation effect of HTBS (A), N. oculata (B), C. sorokiniana (C) under different dose ammonia (/L) and time. Removal efﬁciency of HTBS (D), N. oculata (E), C. sorokiniana (F) after different combination treatment of ammonia adjusting pH and medium, the pH of D–F was 10.8, 10.7, 10.0, respectively. +/pH normal (C): the normal algae medium with/without adjusting corresponding pH as control group;+pH/dH2O (f/2, BBM) (S): the normal algae medium was centrifuged then adjusting corresponding pH after resuspending with dH2O, f/2 or BBM. prepare for SEM, small amounts of ﬂocs from ﬂocculation of algae cells were simply placed on glass slides and air dried to avoid damaging the ﬂocs structure. Dried samples were mounted on copper stubs and sputter coated with gold-palladium. The specimens were observed at 5 kV(Yan et al., 2009). and the bubbling was stopped when the pH was reduced to 6.5. After that the medium was placed on magnetic stirrer and stirred intensely, and the stirring stopped when the pH became stable. 2.4. pH kinetics changes of culture medium and ammonia conversion using CO2 To further analyze the three algae metabolites such as protein and lipid to evaluate whether they were affected by the ammonia induced ﬂocculation, comparisons between ﬂocculation and centrifugation were made. The algae biomasses were harvested using ammonia ﬂocculation and centrifugation, and the contents of neutral lipid, total protein, and pigments were measured. The supernatant of ﬂocculated culture medium was bubbled with pure CO2 using airstones, the velocity of CO2 was controlled at 60 ml/min. The pH was monitored and recorded over time, 2.5. Measurement of protein, pigment and lipid content 301 F. Chen et al. / Bioresource Technology 121 (2012) 298–303 Algal total lipid was extracted according to the procedure reported (Bligh and Dyer, 1959). For each 40 mg of sample, 6 ml of CHCl3: methanol (2:1, v/v) was added and vortexed well, then 2 ml of methanol was added, the supernatant was mixed with 3.6 ml 5% NaCl, then centrifuged at 3000 g for 10 min, ﬁnally the organic phase (bottom) was collected and dried at 60 °C using pressured gas blowing concentrators, the crude oil was weighted. Proteins were extracted using 0.5 M sodium hydroxide followed by boiling 10 min, and then centrifuged at 8000 g for 5 min, the supernatant was measured using Bradford assay (Bradford, 1976). The samples were both centrifuged at 5000 g for 10 min, the pellets were homogenized in an ice-cold 80% acetone (v/v) with a chilled mortar and pestle. The chlorophyll content was determined spectrophotometrically as described in (Arnon, 1949). 2.6. Reuse of ﬂocculated algal culture medium for subsequent cultivation of algae The algae HTBS was cultivated for 16 days at 26 °C using f/2 as the growth medium. The algae were then ﬂocculated using the ammonia-based process, ﬂocs from the culture medium were separated using gravity and centrifuged. The supernatant culture medium was treated with boiling, bubbling with 60 ml/min pure CO2 until pH recovery to about 8.0, combining of bubbling and heat, respectively. After that trace metal and A5 were added before inoculation with a 10% (v/v) HTBS. Sampling was conducted every 2 days. The cultivation and harvest cycles were repeated three times to investigate the growth supportability of the ﬂocculated medium, which was measured based on the spectrophotometer method. In a control experiment, fresh and sterilized f/2 with 1 mM ammonium bicarbonate (NH4HCO3) was used as extra nitrogen and carbon source. 3. Results and discussion 3.1. Analysis of ﬂocculation efﬁciency The effectiveness of the ﬂocculation process appeared very obvious based on the observation of the color change. Supplementa1 Fig. 1A and 1B show the color difference between the untreated and treated algae HTBS. After the addition of aqueous ammonia to the medium and a short-time settling, the upper portion of the medium was highly transparent. The bottom portion of the medium, on the other hand, existed a lot of distinct ﬂocs which were deep green, compact and irregular surface shape. The similar results were also observed with N. oculata and C. sorokiniana. The above observation can be further conﬁrmed with the calculated ﬂocculation efﬁciency. The removal efﬁciency of marine algae HTBS was 66.9% at 38.37 mmol L1 of ammonia within 0.5 h, to 91.2% at 3 h, and to more than 95% at 12 h (Fig. 2 A). The similar results were also obtained when using other two algae strains (Fig. 2 B and C), the settling of N. oculata became substantial when the ammonia concentration was at more than 57.31 mmol L1, the corresponding removal efﬁciency reached to 70.7% within 0.5 h, to 93% at 3 h, and to more than 99% at 12 h. For C. sorokiniana, the trends of the ﬂocculation effect were similar to the marine algae, but the removal efﬁciency was less distinct than marine algae. It was only 16.4% at 113.3 mmol L1 within half an hour, even at 12 h, it just reached to 49.9%. However, the pH of HTBS adding 38.37 mmol ammonia perlitre medium was 10.8; the pH of N. oculata adding 57.31 mmol was 10.7; whereas the pH of C. sorokiniana adding 113.3 mmol was 10.0. Therefore, it suggests that this ﬂocculation process has a certain pH threshold above which it becomes more effective. This is consistent with Vandamme’s study (Vandamme et al., 2012). The pH effect is more sensitive for marine algae than Table 1 Protein contents of three algae strains under centrifugation and aqueous ammonia harvest. Proteins (lg/ml) Aqueous ammonia Centrifugation HTBS C. sorokiniana N. oculata 628 ± 149 286.4 ± 169 295.6 ± 105 626 ± 105 294.8 ± 99 305.6 ± 109 Table 2 Chlorophyll contents of three algae strains under centrifugation and aqueous ammonia harvest. Pigment (lg/ml) HTBS C. sorokiniana N. oculata Chlorophyll Chlorophyll Chlorophyll Chlorophyll Chlorophyll Chlorophyll Chlorophyll Chlorophyll Chlorophyll a b (a + b) a b (a + b) a b (a + b) Aqueous ammonia Centrifugation 19.24 ± 1.562 3.24 ± 1.092 22.48 ± 2.655 10.39 ± 0.171 2.97 ± 0.115 13.36 ± 0.286 8.54 ± 0.314 1.65 ± 0.108 10.19 ± 0.422 16.59 ± 1.243 5.25 ± 0.163 21.84 ± 1.080 11.01 ± 0.275 3.69 ± 0.115 14.70 ± 0.390 8.96 ± 0.105 2.46 ± 0.196 11.42 ± 0.301 Table 3 Lipid contents of three algae strains under centrifugation and aqueous ammonia harvest. Lipid (w/dry) Aqueous ammonia (%) Centrifugation (%) HTBS C. sorokiniana N. oculata 20.6 ± 0.549 23.6 ± 0.054 67.2 ± 1.151 21.1 ± 0.348 23.7 ± 0.026 68.6 ± 1.09 for fresh water algae. It was inferred that the compositions of sea water are complex and some ions react with each other under high pH condition to cause chemical formations that subsequently lead the algal cell surface change to ﬂocculate, which accelerated the settling of the algae. The function of ions as co-factors was further veriﬁed by the results of the experiments testing the ﬂocculation effect (Fig. 2D–F). Regardless the types of culture medium used, the effect of adjusting pH on ﬂocculation performance was remarkable (>99%). However, the removal efﬁciency difference caused by adjusting pH in distilled water (dH2O) was not at all signiﬁcant (<5%). This demonstrates that dramatically ﬂocculation effects cannot be obtained by only increasing pH. These results are in agreement with previous studies (Sukenik and Shelef, 1983; Vandamme et al., 2012). The pH-related coagulation and ﬂocculation mechanisms were believed to be affected by the balance between the electrostatic repulsion and the Van der Waals attraction through increasing ionic strength in medium, reducing the microalgae surface charge or being charge neutralization (Vandamme et al., 2012). In contrast, the results of this study suggest that ﬂocculation of algae is caused by another mechanism: the rising pH generates magnesium hydroxide, calcium hydroxide (Vandamme et al., 2012), calcium phosphate (Sukenik and Shelef, 1983), and other insoluble compound particles which covered the algae cell surface (Supplemental Fig. 1D), which in combination with charge neutralization or adhesive attraction, forming heavier coagulation ﬂocs and settling. Therefore, using the aqueous ammonia ﬂocculation process to harvest algae was more suitable for marine algae applications. 3.2. SEM analysis The results of SEM analysis showed that cell morphology was fuzzy, ﬂagella shedded, surface damaged. Cells adhesion occurred with a large number of coat (Supplemental Fig. 1D). The normal 302 F. Chen et al. / Bioresource Technology 121 (2012) 298–303 were also observed (Tables 2 and 3). These results therefore suggest that ﬂocculation harvesting using aqueous ammonia rarely inﬂuenced the changes of algae metabolite contents. The similar results were also reported by other researchers (Knuckey et al., 2006; Vandamme et al., 2012). 3.4. Reuse of ﬂocculated algal culture medium for cultivation of algae Fig. 3. Reuse of ﬂocculated supernatant using aqueous ammonia. The medium was treated with CO2 (square), heat (circle), combination of heat and CO2 (up triangular), respectively, NH4HCO3 (down triangular) as control. algae shape however, was clear, no damaged, recognizable ﬂagella, no excess ﬂoc cover, with dispersed cell particles (Supplemental Fig. 1C). This explains the reason that allows the cells to settle. Microalgae are generally small (1–30 lm) with low density (i.e. similar to culture medium) and the surface is with negative charge. These factors make the microalgae not ﬂocculate under normal growth conditions. However, after adding aqueous ammonia, the surface structure reacted with ammonia and was damaged, ﬁnally formed lumps by incorporating other ions. That was equivalent to increase the volume and cell density, which was higher than the density of medium, causing sedimentation to occur. The speciﬁc components of these coverings or adhesion complexes, however, are still unclear. More research is required to investigate the components of ﬂocs after precipitation of algae occurs. 3.3. Metabolite inﬂuence of algae after ﬂocculation using aqueous ammonia The results showed that the total protein content of the HTBS, C.sorokiniana, N. oculata harvested using centrifugation was 0.626, 0.294, 0.305 mg/ml, respectively, whereas that using aqueous ammonia was 0.628, 0.286, 0.295 mg/ml, respectively (Table 1). These results indicate that the protein content difference between ammonia ﬂocculation and centrifugation is not signiﬁcant (p < 0.05). The similar results on chlorophyll and lipid contents The HTBS algae strains were used to evaluate the suitability of the recycled culture medium for new culture. The supernatant of growing medium was simply treated by method described earlier (2.6). The results showed that algae HTBS could normally grow in the presence of 1 mM NH4HCO3, the OD600 increased 3.4 times after 6 days (Fig. 3), indicating that algae can utilize not only NH4+, but also HCO3-. But algae were not good growth only treated by bubbling CO2 or heating. Although Conover (1975) observed that algae could assimilate not only nitrate but also urea and ammonium, ammonia inhibited photosynthesis and growth of Scenedesmus obliquus at concentrations over 2.0 mM and at pH values over 8.0, other algae such as Chlorella pyrenoidosa, Anacystis nidulans, and Plectonema boryanum were also susceptible to ammonia inhibition (Aharon and Yosef, 1976). However, the OD600 of algae under combination of heating and bubbling CO2 grew two times after 6 days. Combining heating and bubbling CO2, on the one hand, might have stripped out part of the ammonia, which was similar with Roney’s results. The concentration of NH3 was mostly dependent on temperature, the solubility of NH3 in water could decrease to 18% (w/ w) at 50 °C from 47% (w/w) at 0 °C (Roney et al., 2004). At the same time, CO2 addition helped to convert the residual ammonia into ammonium. The amount of CO2 required to bring back the pH to 8.0 (proper for marine algae) was also estimated (Fig. 4) according to the equilibrium NH3 + H2O ¡ NH3H2O ¡ NH4+ + OH (Nickolette et al., 2004), and the rate of CO2 outgassing through an air– water surface to the atmosphere (Chi et al., 2011; Zeebe and Wolf-Gladrow, 2001). With assumed conditions of 20 mM inorganic carbon, the transfer coefﬁcient which was 1 106 m/s, the amount of CO2 required was approximately 38.37 mM to lower the pH from 10.8 to 8.0 for HTBS culture. Improving the efﬁciency of conversion of ammonia and carbon dioxide, however, needs further to be researched. 4. Conclusions The novel process using ammonia to ﬂocculate algae is effective for the tested different algae strains. The culture medium after the treatment can be reused for algae culture. Ammonia as a ﬂocculant without metal ions contamination also does not change the algae Fig. 4. pH kinetic curve of ﬂocculated supernate using aqueous ammonia with CO2 (A) and stirred after stopping bubbling CO2 (B). F. Chen et al. / Bioresource Technology 121 (2012) 298–303 cell metabolic components. Further research needs to be conducted reveal the speciﬁc mechanism and to optimize the process. Acknowledgements This work was supported partially by the National Program on Key Basic Research Project (2011CB200905 and 2011CB200906) and by Tianjin Municipal Science and Technology special project (No. 10ZCKFSY05400), the Hi-Tech Research and Development Program (863) of China (2012AA052103), science and technology support key project plan of Tianjin (12ZCZDSFO2000). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2012. 06.076. References Aharon, A., Yosef, A., 1976. 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