Bioresource Technology 121 (2012) 298–303

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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 a b s t r a c t

Article history: Microalgae have been considered as a promising feedstock for biofuels and greenhouse gas reduction. A
Received 7 May 2012 low-cost harvesting technology without secondary contamination for down-stream extraction is a key
Received in revised form 26 June 2012 requirement to make algal biofuel commercially viable. A novel harvesting method using ammonia as
Accepted 27 June 2012
a flocculant to make the algal biomass settable was devised and studied. Another major advantage of this
Available online 3 July 2012
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
Keywords:
of algae growing in the ammonia-enriched flocculation medium treated with heating and CO2 was 2
Aqueous ammonia
Algae
times than that of initial after 6 days. These results suggested that this flocculation method was efficient,
Harvest convenient and allowed the reuse of the flocculated medium, therefore providing an option for economic
Reuse harvesting and cultivation of microalgae.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction cultivation of microalgae has been widely regarded as one of the

most promising alternatives that have potential to deal with the
With the continuous depletion of petroleum and the rising CO2 problem of global warming (Benemann and Oswald, 1996; Chisti,
concentration in the atmosphere from the combustion of fossil fuels, 2007).
increasing attention has been given to the development of the alter- Many technical barriers, however, exist to the commercializa-
native fuels, such as lipid-derive biodiesel (Iglesias-Rodriguez, tion of algal biofuel systems among which concentrating the algal
2008) and renewable hydrocarbons (Graves et al., 2011). Biodiesel biomass is a major challenge (Wang et al., 2008). It was estimated
has traditionally been produced with a variety of feedstocks such that the cost of harvesting algal biomass was 20–30% of the total
as soybeans, canola oil, animal fat, palm oil, corn oil, waste cooking costs of production (Gudin and Thepenier, 1986) due to small cell
oil (Felizardo et al., 2006), jatropha oil (Barnwal and Sharma, 2005). size (typically in the range of 1–30 lm), low concentration (typi-
These conventional lipid sources not only are subjected to quantity cally in the range of 0.3–5 g/l), unfavourable density (only slightly
limitation, but also cause concerns in competition with the need as greater than media) and negative charge (contributing to their sta-
food for human. Algae have attracted a great interest in recent years bility in a dispersed state) (Brennan and Owende, 2010).
because of their various desirable characteristics such as high Major techniques available for harvesting microalgae include
potential productivity (Amaro et al., 2011; Brennan and Owende, filtration, centrifugation, gravity sedimentation, flocculation
2010; Chisti, 2007). Additionally, algae can be potentially produced (Benemann and Oswald, 1996; Molina et al., 2003) and electropho-
with resources such as salt water or wastewater that are not used for resis (Amaro et al., 2011; Danquah et al., 2009). However, rapid
food production. Moreover, biofuel production from large-scale clogging, continuous backwashing and the costs arising from
pumping and membrane replacement are the major problems in
⇑ Corresponding author. filtration techniques (Becker, 1994; Molina et al., 2003). Centrifu-
E-mail address: chen_sl@[Link] (S. Chen). gation requires high energy input, complicated processing and

0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
[Link]
 F. Chen et al. / Bioresource Technology 121 (2012) 298–303 299

large capital investment (Benemann and Oswald, 1996; Molina (fresh water), Nannochlropsis oculata (marine), and native algal
et al., 2003). Gravity settling is suitable only to harvest large-sized species (marine, named HTBS, classified as Dunaliella). C. sorokini-
microalgal cells, e.g. Spirulina spp.(Xiong et al., 2008). Electrolytic ana was cultivated using a Bold’s Basal Medium without glucose
processes with increasing system temperature and cathode fouling (Bischoff and Bold, 1963), the pH of the medium was adjusted to
lead to high power consumption (Amaro et al., 2011). 6.1 before sterilization. The marine algae were cultivated using a
The harvesting of microalgal cells by flocculation is seen to be a f/2 medium (Guillard and Ryther, 1962) with filtered sea water,
superior method to other aforementioned harvesting methods be- the composition briefly was: 1.5 g NaNO3, 0.04 g K2HPO4, 0.006 g
cause of its effectiveness with given cost (Pushparaj et al., 1993). ferric ammonium citrate, and trace metal mix A5 in 1 L of distilled
Chemicals called flocculants are usually added to induce flocculation water without adjusting pH (pH was 8.0). The strains were incu-
followed by gravity separation. Multivalent metal salts like ferric bated in plat bioreactor that contained 15 L of the f/2 medium. The
chloride (FeCl3), aluminium sulphate (Al2(SO4)3) and ferric sulphate algal culture was continuously bubbling sterilized air with 3% CO2
(Fe2(SO4)3) (Shelef et al., 1984) and certain cationic polymers such under continuous illumination at 150 lmol m2 s1. The culture
as, chitosan, cationic polyacrlyamides, and cellulose, surfactants, temperature was 25 ± 1 °C and the strain was cultivated for
and other man-made fibers (Bilanovic et al., 1988; Oh et al., 2001; 14 days.
Pushparaj et al., 1993) have been tested effective. Although floccula-
tion has proven to be successful for concentrating microalgae, a large 2.2. Experimental design and analysis of flocculation efficiency
amount of flocculant is needed to cause solid–liquid separation of
the microalgae. The algal biomass as the end product is contami- In order to assess the flocculation effect of aqueous ammonia,
nated by the added flocculant, thus algae harvesting with chemical the experiment of HTBS comparing with HTBS-treated algae with
flocculant is not suitable for biofuel application due the added cost aqueous ammonia was first carried out. To further analyze the floc-
and residual effects on algal biomass and the culture water. culation effect quantitatively, the aforementioned three strains
An ideal flocculant for microalgae harvesting must meet the fol- were tested. The cultures were stirred using magnetic stirrer and
lowing criteria: (i) resulting in no residual in biomass, (ii) leading added different doses of commercial aqueous ammonia by titrime-
to high efficient subsequent settling of aglae, (iii) allowing reusing try. For HTBS the concentrations were 0.09, 0.36, 1.09, 3.12, 12.90,
the algal culture medium as growth supporting nutrients for sub- 38.37 mmol L1, For N. oculata the concentrations were 0.74, 2.22,
sequent algal cultivation, (iv) considering the environmental im- 4.44, 10.72, 26.62, 57.31, 118.7 mmol L1, and for C. sorokiniana
pact, reducing the greenhouse gas effect. A flocculation process the concentrations were 0.52, 4.95, 9.54, 24.26, 48.14, 113.3,
using ammonia as coagulant was developed to meet these criteria. 240.6 mmol L1. After the ammonia addition, the removal efficiency
In this process, aqueous ammonia is used to alter the pH of the cul- of flocculated algal cultures was measured at 0.5 h, 1 h, 3 h, 6 h,
ture that leads to the flocculation and settling of the algae. Upon 12 h. An aliquot was taken at a height of two-thirds from the bot-
the removal of the algal biomass, flue gas containing CO2 is intro- tom, and the pH was measured. The optical density (OD) of the ali-
duced to lower the pH to convert the un-ionized ammonia to its io- quot was measured at 600 nm to evaluate the flocculation efficiency
nic form to reduce its potential toxicity when the ammonia laden of the aqueous ammonia (Kim et al., 2011). The flocculating effi-
water is resued for algae culture (Fig. 1). This paper presents the ciency was calculated using the following equation:
test results of this process, including flocculation efficiency, ammo-
nia conversion using CO2, analysis of cell metabolite and morphol- Flocculating efficiency ð%Þ ¼ ð1  B=AÞ  100
ogy, and reuse of the flocculated culture water. Upon further
where, A is the optical density of the algal culture before the floccu-
refinement, this process has a potential to provide a viable option
lation measured at 600 nm and B is the optical density of the sam-
for bio-friendly and economic mass cultivation and harvesting of
ple at 600 nm. The supernatant of culture medium was used to
microalgae to make algal biofuel production more competitive.
carry out reuse experiment.

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 ammonia-based flocculation process was evaluated with the flocculation experiment. HTBS algae cells were placed on glass
both freshwater and marine algae, including Chlorella sorokiniana slides and dried in air. Instead of fixation as traditionally used to

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 and the bubbling was stopped when the pH was reduced to 6.5.
cells were simply placed on glass slides and air dried to avoid dam- After that the medium was placed on magnetic stirrer and stirred
aging the flocs structure. Dried samples were mounted on copper intensely, and the stirring stopped when the pH became stable.
stubs and sputter coated with gold-palladium. The specimens were
observed at 5 kV(Yan et al., 2009). 2.5. Measurement of protein, pigment and lipid content

2.4. pH kinetics changes of culture medium and ammonia conversion To further analyze the three algae metabolites such as protein
using CO2 and lipid to evaluate whether they were affected by the ammonia
induced flocculation, comparisons between flocculation and cen-
The supernatant of flocculated culture medium was bubbled trifugation were made. The algae biomasses were harvested using
with pure CO2 using airstones, the velocity of CO2 was controlled ammonia flocculation and centrifugation, and the contents of neu-
at 60 ml/min. The pH was monitored and recorded over time, tral lipid, total protein, and pigments were measured.
 F. Chen et al. / Bioresource Technology 121 (2012) 298–303 301

Algal total lipid was extracted according to the procedure re- Table 1
ported (Bligh and Dyer, 1959). For each 40 mg of sample, 6 ml of Protein contents of three algae strains under centrifugation and aqueous ammonia
harvest.
CHCl3: methanol (2:1, v/v) was added and vortexed well, then
2 ml of methanol was added, the supernatant was mixed with Proteins (lg/ml) Aqueous ammonia Centrifugation
3.6 ml 5% NaCl, then centrifuged at 3000  g for 10 min, finally HTBS 628 ± 149 626 ± 105
the organic phase (bottom) was collected and dried at 60 °C using C. sorokiniana 286.4 ± 169 294.8 ± 99
pressured gas blowing concentrators, the crude oil was weighted. N. oculata 295.6 ± 105 305.6 ± 109

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). Table 2
The samples were both centrifuged at 5000  g for 10 min, the Chlorophyll contents of three algae strains under centrifugation and aqueous
pellets were homogenized in an ice-cold 80% acetone (v/v) with a ammonia harvest.
chilled mortar and pestle. The chlorophyll content was determined Pigment (lg/ml) Aqueous ammonia Centrifugation
spectrophotometrically as described in (Arnon, 1949).
HTBS Chlorophyll a 19.24 ± 1.562 16.59 ± 1.243
Chlorophyll b 3.24 ± 1.092 5.25 ± 0.163
Chlorophyll (a + b) 22.48 ± 2.655 21.84 ± 1.080
2.6. Reuse of flocculated algal culture medium for subsequent
C. sorokiniana Chlorophyll a 10.39 ± 0.171 11.01 ± 0.275
cultivation of algae Chlorophyll b 2.97 ± 0.115 3.69 ± 0.115
Chlorophyll (a + b) 13.36 ± 0.286 14.70 ± 0.390
The algae HTBS was cultivated for 16 days at 26 °C using f/2 as N. oculata Chlorophyll a 8.54 ± 0.314 8.96 ± 0.105
the growth medium. The algae were then flocculated using the Chlorophyll b 1.65 ± 0.108 2.46 ± 0.196
Chlorophyll (a + b) 10.19 ± 0.422 11.42 ± 0.301
ammonia-based process, flocs from the culture medium were sep-
arated using gravity and centrifuged. The supernatant culture med-
ium was treated with boiling, bubbling with 60 ml/min pure CO2
until pH recovery to about 8.0, combining of bubbling and heat, Table 3
respectively. After that trace metal and A5 were added before inoc- Lipid contents of three algae strains under centrifugation and aqueous ammonia
harvest.
ulation with a 10% (v/v) HTBS. Sampling was conducted every
2 days. The cultivation and harvest cycles were repeated three Lipid (w/dry) Aqueous ammonia (%) Centrifugation (%)
times to investigate the growth supportability of the flocculated HTBS 20.6 ± 0.549 21.1 ± 0.348
medium, which was measured based on the spectrophotometer C. sorokiniana 23.6 ± 0.054 23.7 ± 0.026
method. In a control experiment, fresh and sterilized f/2 with N. oculata 67.2 ± 1.151 68.6 ± 1.09
1 mM ammonium bicarbonate (NH4HCO3) was used as extra nitro-
gen and carbon source.
for fresh water algae. It was inferred that the compositions of sea
water are complex and some ions react with each other under high
3. Results and discussion
pH condition to cause chemical formations that subsequently lead
the algal cell surface change to flocculate, which accelerated the set-
3.1. Analysis of flocculation efficiency
tling of the algae. The function of ions as co-factors was further ver-
ified by the results of the experiments testing the flocculation effect
The effectiveness of the flocculation process appeared very
(Fig. 2D–F). Regardless the types of culture medium used, the effect
obvious based on the observation of the color change. Supple-
of adjusting pH on flocculation performance was remarkable
menta1 Fig. 1A and 1B show the color difference between the un-
(>99%). However, the removal efficiency difference caused by
treated and treated algae HTBS. After the addition of aqueous
adjusting pH in distilled water (dH2O) was not at all significant
ammonia to the medium and a short-time settling, the upper por-
(<5%). This demonstrates that dramatically flocculation effects
tion of the medium was highly transparent. The bottom portion of
cannot be obtained by only increasing pH. These results are in
the medium, on the other hand, existed a lot of distinct flocs which
agreement with previous studies (Sukenik and Shelef, 1983; Vand-
were deep green, compact and irregular surface shape. The similar
results were also observed with N. oculata and C. sorokiniana. amme et al., 2012). The pH-related coagulation and flocculation
mechanisms were believed to be affected by the balance between
The above observation can be further confirmed with the calcu-
the electrostatic repulsion and the Van der Waals attraction through
lated flocculation efficiency. The removal efficiency of marine algae
increasing ionic strength in medium, reducing the microalgae sur-
HTBS was 66.9% at 38.37 mmol L1 of ammonia within 0.5 h, to
face charge or being charge neutralization (Vandamme et al.,
91.2% at 3 h, and to more than 95% at 12 h (Fig. 2 A). The similar re-
2012). In contrast, the results of this study suggest that flocculation
sults were also obtained when using other two algae strains (Fig. 2 B
of algae is caused by another mechanism: the rising pH generates
and C), the settling of N. oculata became substantial when the
magnesium hydroxide, calcium hydroxide (Vandamme et al.,
ammonia concentration was at more than 57.31 mmol L1, the cor-
2012), calcium phosphate (Sukenik and Shelef, 1983), and other
responding removal efficiency reached to 70.7% within 0.5 h, to 93%
insoluble compound particles which covered the algae cell surface
at 3 h, and to more than 99% at 12 h. For C. sorokiniana, the trends of
(Supplemental Fig. 1D), which in combination with charge neutral-
the flocculation effect were similar to the marine algae, but the re-
ization or adhesive attraction, forming heavier coagulation flocs and
moval efficiency was less distinct than marine algae. It was only
settling. Therefore, using the aqueous ammonia flocculation process
16.4% at 113.3 mmol L1 within half an hour, even at 12 h, it just
to harvest algae was more suitable for marine algae applications.
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 3.2. SEM analysis
113.3 mmol was 10.0. Therefore, it suggests that this flocculation
process has a certain pH threshold above which it becomes more The results of SEM analysis showed that cell morphology was
effective. This is consistent with Vandamme’s study (Vandamme fuzzy, flagella shedded, surface damaged. Cells adhesion occurred
et al., 2012). The pH effect is more sensitive for marine algae than 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 sug-
gest 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

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 bub-
bling CO2 or heating. Although Conover (1975) observed that algae
could assimilate not only nitrate but also urea and ammonium,
Fig. 3. Reuse of flocculated supernatant using aqueous ammonia. The medium was ammonia inhibited photosynthesis and growth of Scenedesmus obli-
treated with CO2 (square), heat (circle), combination of heat and CO2 (up quus at concentrations over 2.0 mM and at pH values over 8.0, other
triangular), respectively, NH4HCO3 (down triangular) as control. algae such as Chlorella pyrenoidosa, Anacystis nidulans, and Plecto-
nema boryanum were also susceptible to ammonia inhibition
(Aharon and Yosef, 1976). However, the OD600 of algae under com-
algae shape however, was clear, no damaged, recognizable flagella,
bination of heating and bubbling CO2 grew two times after 6 days.
no excess floc cover, with dispersed cell particles (Supplemental
Combining heating and bubbling CO2, on the one hand, might have
Fig. 1C). This explains the reason that allows the cells to settle. Mic-
stripped out part of the ammonia, which was similar with Roney’s
roalgae are generally small (1–30 lm) with low density (i.e. similar
results. The concentration of NH3 was mostly dependent on tem-
to culture medium) and the surface is with negative charge. These
perature, the solubility of NH3 in water could decrease to 18% (w/
factors make the microalgae not flocculate under normal growth
w) at 50 °C from 47% (w/w) at 0 °C (Roney et al., 2004). At the same
conditions. However, after adding aqueous ammonia, the surface
time, CO2 addition helped to convert the residual ammonia into
structure reacted with ammonia and was damaged, finally formed
ammonium. The amount of CO2 required to bring back the pH to
lumps by incorporating other ions. That was equivalent to increase
8.0 (proper for marine algae) was also estimated (Fig. 4) according
the volume and cell density, which was higher than the density of
to the equilibrium NH3 + H2O ¡ NH3H2O ¡ NH4+ + OH (Nicko-
medium, causing sedimentation to occur. The specific components
lette et al., 2004), and the rate of CO2 outgassing through an air–
of these coverings or adhesion complexes, however, are still un-
water surface to the atmosphere (Chi et al., 2011; Zeebe and
clear. More research is required to investigate the components of
Wolf-Gladrow, 2001). With assumed conditions of 20 mM inor-
flocs after precipitation of algae occurs.
ganic carbon, the transfer coefficient which was 1  106 m/s, the
amount of CO2 required was approximately 38.37 mM to lower
3.3. Metabolite influence of algae after flocculation using aqueous the pH from 10.8 to 8.0 for HTBS culture. Improving the efficiency
ammonia of conversion of ammonia and carbon dioxide, however, needs fur-
ther to be researched.
The results showed that the total protein content of the HTBS,
[Link], N. oculata harvested using centrifugation was 4. Conclusions
0.626, 0.294, 0.305 mg/ml, respectively, whereas that using aque-
ous ammonia was 0.628, 0.286, 0.295 mg/ml, respectively (Table The novel process using ammonia to flocculate algae is effective
1). These results indicate that the protein content difference be- for the tested different algae strains. The culture medium after the
tween ammonia flocculation and centrifugation is not significant treatment can be reused for algae culture. Ammonia as a flocculant
(p < 0.05). The similar results on chlorophyll and lipid contents 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) 298–303 303

cell metabolic components. Further research needs to be con- Danquah, M., Ang, L., Uduman, N., Moheimani, N., Fordea, G., 2009. Dewatering of
microalgal culture for biodiesel production: exploring polymer flocculation and
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