Bioresource Technology 221 (2016) 385393

Contents lists available at ScienceDirect

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

Nitrogen and phosphorus removal coupled with carbohydrate

production by five microalgae cultures cultivated in biogas slurry
Fen Tan a, Zhi Wang a, Siyu Zhouyang a, Heng Li b, Youping Xie a, Yuanpeng Wang a, Yanmei Zheng a,
Qingbiao Li a,b,
a
Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University,
Xiamen 361005, PR China
b
College of the Environment and Ecology, Xiamen University, Xiamen 361005, PR China

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Production of carbohydrate in Graphic Abstract

microalgae from biogas slurry was
proven feasible. Light CO2

High ammonia removal efficiency,
rate and carbohydrate content have Carbohydrate
been obtained in biogas slurry. 61.5%
Content

Phosphorus and magnesium P Mg

starvation is proposed as a valid
strategy for advancing the timing of
carbohydrate accumulation in biogas N/P depletion
biogas slurry
slurry. Limitation
Diluted biogas

Magnesium is proved firstly as the
slurry
influence factor for carbohydrate
accumulation.

a r t i c l e i n f o a b s t r a c t

Article history: In this study, five microalgae strains were cultured for their ability to survive in biogas slurry, remove
Received 4 July 2016 nitrogen resources and accumulate carbohydrates. It was proved that five microalgae strains adapted
Received in revised form 6 September 2016 in biogas slurry well without ammonia inhibition. Among them, Chlorella vulgaris ESP-6 showed the best
Accepted 7 September 2016
performance on carbohydrate accumulation, giving the highest carbohydrate content of 61.5% in biogas
Available online 13 September 2016
slurry and the highest ammonia removal efficiency and rate of 96.3% and 91.7 mg/L/d respectively in
biogas slurry with phosphorus and magnesium added. Additionally, the absence of phosphorus and mag-
Keywords:
nesium that can be adverse for biomass accumulation resulted in earlier timing of carbohydrate accumu-
Biogas slurry
Ammonia nitrogen uptake
lation and magnesium was firstly recognized and proved as the influence factor for carbohydrate
Carbohydrate accumulation accumulation. Microalgae that cultured in biogas slurry accumulated more carbohydrate in cell, making
Chlorella vulgaris biogas slurry more suitable medium for the improvement of carbohydrate content, thus can be regarded
as a new strategy to accumulate carbohydrate.
2016 Elsevier Ltd. All rights reserved.

1. Introduction

Anaerobic digestion is widely applied in clean energy produc-

Corresponding author at: Department of Chemical and Biochemical Engineer-
ing, College of Chemistry and Chemical Engineering, Xiamen University, No. 422,
Siming South Road, Xiamen 361005, PR China.
E-mail address: kelqb@xmu.edu.cn (Q. Li).
tion, solving the problem of waste contamination. Associate with
the development of anaerobic digestion engineering, the increasing
output of biogas slurries becomes inevitable and approximately
385 million tons of liquid waste was generated by over 30 million

http://dx.doi.org/10.1016/j.biortech.2016.09.030
0960-8524/ 2016 Elsevier Ltd. All rights reserved.
386 F. Tan et al. / Bioresource Technology 221 (2016) 385393

methane-generating tanks (Huang et al., 2014). With abundant 2. Materials and methods
nitrogen resources as a leading cause of eutrophication in the slur-
ries, draining biogas slurries directly to the environment without 2.1. Source of biogas slurry
sufficient pretreatment is considered to be at high risk of environ-
mental hazards. Meanwhile, biogas slurry is particularly rich in Biogas slurry used in this paper was obtained from the outlet of
nitrogen resources which are exactly needed for microalgae a 10 L semi-continuously fed swine anaerobic digester which has
growth. Culturing microalgae to utilize nutrients in the bio- 400 mL daily import (TS = 10%) and output. Biogas slurry was the
slurries and to produce high value-added products have been com- supernatant liquid of output which was centrifuged (universal
monly realized as a potential strategy for emission reduction and 320R, Hettich, Germany) in 8000 rpm for 10 min. Biogas slurry col-
renewable energy production (Lam and Lee, 2012). Among the lected daily and stored in a 4 C refrigerator without autoclaving or
microalgae-based high-value products, carbohydrate is one of the adjusting of pH value. However, the initial concentration of ammo-
bio-products and potential feedstock for bioethanol production nia in biogas slurry is too high for microalgae strains to bear (Tam
which has provoked heightened interest since energy crisis and Wong, 1996). Meanwhile, the color of the biogas slurry is deep
(Maity et al., 2014). dark brown which would adversely affect light penetration and
To improve microalgae-based carbohydrate production, therefore prevent algal growth. Dilution of biogas slurry based on
researches have been designed and conducted. It is generally the ammonia concentration thus needed for the culture of microal-
believed that carbohydrate generating process happened in Calvin gae and 8 times diluted biogas slurry was employed in this study
cycle of photosynthesis and nitrogen starvation is one of the most (Table 1).
effective way to trigger carbohydrate accumulation (Markou et al.,
2015). Yet culturing microalgae in pure medium to produce carbo-
2.2. Microalgae strains and preculture conditions
hydrate as the feedstock of bio-ethanol is less competitive to tradi-
tional fossil energy and competed for freshwater (Ho et al., 2013).
C. sorokiniana FACHB-275, C. vulgaris and S. dimorphus FACHB-
Limited researches have been conducted with carbohydrate
1266 were bought from algal-species database of Wuhan Institute
production as target in wastewater, among which Spirulina sub-
of Hydrobiology, Chinese Academy of Sciences. C. vulgaris ESP-6
salsa, Scenedesmus obliquus and Chlorella vulgaris have been cul-
and Desmodesmus sp. F51 were obtained from the Department of
tured in waste water of monosodium glutamate factory, food and
Chemical Engineering, National Cheng Kung University, Taiwan.
municipal wastewater treatment plant respectively, achieving the
All the microalgae species used in this study have been tested in
highest carbohydrate content of 50% (He et al., 2013; Ji et al.,
previous researches that conducted in waste water (Quiroz Arita
2015; Jiang et al., 2015), proving the feasibility of microalgae-
et al., 2015), proving a good potential and feasibility of apply these
based carbohydrate production in wastewater. However, distin-
strains to biogas slurry. Microalgae strains were precultured at
guish from municipal wastewater and industrial wastewater, bio-
27 1 C in 1 L Modified Bold Basal 3N medium (Modified BBM)
gas slurry is recognized to have higher ammonia concentration
which consists of (g/L) NaNO3 (0.075), K2HPO4 (0.0383), KH2PO4
and turbidity with low useable carbon resource and only quite a
(0.088), MgSO47H2O (0.075), CaCl22H2O (0.025), NaCl (0.025),
few studies using biogas slurry as culture media for biogas slurry
FeCl36H2O (0.00177), EDTA (0.00244), trace metal solution consist
treatment, neither did nutrients optimization employed to further
of (mg/L) ZnSO47H2O (0.073), CoSO47H2O (0.016), MnSO45H2O
improve carbohydrate productivity in biogas slurry. Manjinder
(0.584), Na2MnO42H2O (0.00148), NiCl26H2O (0.000149). The
consorted 3 microalgae strains in approximately 26-fold diluted
pre-culture medium was autoclaved at 121 C for 20 min. The
poultry litter anaerobic digestion, achieving the highest carbohy-
microalgae strain culture was illuminated 24 h a day with a light
drate content of 27.3% which is comparatively low (Singh et al.,
intensity of approximately 60 lmol/m2/s. The pre-culture main-
2011). Yue Wang cultivated C. vulgaris JSC-6 with 5-fold diluted
tained about 3.55 days with a continuous supply of 2.5% CO2 at
swine wastewater, achieving carbohydrate content reached up to
an aeration rate of 0.2 vvm and an agitation rate of 300 rpm. The
58% (per dry weight) and yet failed to declined ammonia to emis-
OD685 of microalgae inoculum was about 0.620 after 5-fold diluted
sion standard with above 150 mg/L ammonia left over after
with distilled water. Microalgae strains were maintained in a BG-
12 days cultivation (Wang et al., 2015). With these previous stud-
11 plate for long time storage.
ies reported, it was hard to find a microalga that could accumulate
high carbohydrate and consumed up ammonia quickly in biogas
slurry at the same time. 2.3. Experimental setup
Therefore, two microalgae species (five strains) were employed
for their ability to survive in bio-slurry, remove nitrogen resources Glass bottle (1000 ml) sealed with rubber plug was used as
and accumulate carbohydrates simultaneously in this study. Mean- batch photo bioreactor with external light illuminations provided
while, it has been commonly realized that nitrogen depletion could by fluorescent lamps (14W TWG114, Philips, China) that equipped
enhance the carbohydrate content and nitrogen-starvation strat- along both of its sides (Fig. 1). Three different media were used for
egy has been widely employed to trigger carbohydrate accumula- cultivation of microalga strains in the PBR: Modified Bold Basal 3N
tion, yet no efforts have been made to evaluate whether other
components in the biogas slurry have a positive or negative influ-
Table 1
ence on carbohydrate accumulation. Nutrients such as phosphorus
Biogas slurry nutritional liquid formula.
and magnesium thus were added to further improve the cell den-
sity, ammonia removal performance and carbohydrate accumula- Component Solution (mg/L) Component Solution (mg/L)

tion of three microalgae strains in diluted biogas slurry. By TN 146.64 8.12 TP 2.18
comparing the performance of five microalgae strains in diluted NH4-N 135.5143 PO3
4 -P 2.01 0.04
COD 120.77 15.09 Mg 0.9429
biogas slurry and further medium optimization, a high perfor-
Ca 0.5762 Na 6.8470
mance microalgae strain adapted to the biogas slurry with high K 4.2381 Cu 234.5 * 103
carbohydrate content and high ammonia removal rate can be Cr 4.444 * 103 As 14.95 * 103
selected and utilized as the potential feedstock for bioethanol Zn 642 * 103 Pb 4.897 * 103
production. Cd 1.093 * 103 N:P 73:1
 F. Tan et al. / Bioresource Technology 221 (2016) 385393
Fig. 1. Schematic description photobioreactor.
medium (Modified BBM), diluted biogas slurry and Modified
diluted biogas slurry (added with 0.0383 g/L KH2PO4, 0.088 g/L
K2HPO4 and 0.075 g/L MgSO47H2O). The anaerobic digestion bio-
gas slurry of piggery waste contains over 90% ammonia nitrogen.
However, using NH4-Cl as nitrogen resource in artificial medium
caused serious drop on pH, which started at 7.57 and ended at
3.23 after 24 h, leading microalgae to death. This result corrobo-
rated by related study which explained the sharply decreased pH
was caused by hydrogen ions after ammonia metabolized by
microalgae (Kim et al., 2013). Due to the reason above, five differ-
ent microalgae strains were cultured in Modified BBM, in which
NaNO3 was used as nitrogen resource. After pre-cultivation, algal
seeds were centrifuged at 4000 rpm for 5 min. The batch cultiva-
tion of the microalgae strains was operated at pH value about
7.57.9 under a light intensity of approximately 600 lmol/m2/s.
Temperature, agitation rate and CO2 flow rate were the same as
mentioned in Section 2.2. Samples were collected from the sealed
glass bottle to determine the biomass concentration, pH value, the
residual nitrate concentration, phosphates concentration and car-
bohydrate content in the microalgae cell at set time intervals.
The whole experiments were carried out with duplicates and all
the values in this paper were shown in average values.
2.4. Analytical methods
2.4.1. Determination of microalgae strains biomass concentration
Biomass concentration in Modified BBM was determined by
building up the calibration curve between cell density and OD685
(UV-1800, SHIMADZU, Japan) for each microalgae strain.
While in biogas slurry, biomass concentration was measured
gravimetrically by dry weight to avoid back ground value of biogas
slurry existed at a wavelength of 685 nm. Samples were taken and
centrifuged (4000 rpm, 10 min). The supernatant was removed and
microalgae were collected, washed and centrifuged by deionized
water thrice in order to get rid of impurities completely. The sed-
iments were dried at 80 C until the weight keep constant.
2.4.2. Determination of growth kinetic parameters
The specific growth rate of each microalgae strain was calcu-
lated through biomass concentration and time. The equation is
listed as follows:
LnX 2  LnX 1
R 1
T2  T1
where X represents dry biomass concentrations at day T, 1 and 2
represent different time points.
387
The biomass productivity for five different strains was calcu-
lated by using variation of biomass concentration versus time per-
iod within this variation. The formula is listed as followed:
Xt  X0
P j 2
T  T 0 max
where X represents dry biomass concentrations at day T.
2.4.3. Determination of residual concentration of total nitrogen and
ammonia
The total nitrogen was determined (KDN-12C, TOP, China) by
which H2SO4, CuSO4 and K2SO4 were added with 10 ml sample
for digestion at 420 C for 2 h. When the samples cool down to
room temperature, NaOH (40%) was added and then distillation
begins. Collecting the distillation fraction with a volumetric flask
(250 ml) after which TN was transformed into the form of ammo-
nia nitrogen and determined with UV spectrophotometer. Specific
procedure is described with APHA Standard Methods (APHA,
1998).
The residual concentration of ammonia nitrogen was deter-
mined with UV spectrophotometer at a wavelength of 420 nm.
To get residual concentration of ammonia nitrogen, standard curve
needs to be built firstly. Specific method is described in APHA
Standard Methods (APHA, 1998).
2.4.4. Determination of residual concentration of total phosphorus and
dissolvable phosphorus
The determinations of residual concentration of total phospho-
rus and dissolvable phosphorus were alike in general. Samples
were mixed with H2SO4 and digested at 121 C for 30 min after
which total phosphorus was transformed into dissolvable phos-
phorus and determined with UV spectrophotometer at a wave-
length of 710 nm.
To detect the residual concentration of dissolvable phosphorus,
standard curve needs to be established using K2HPO4. Specific
method is described in APHA Standard Methods (APHA, 1998).
2.4.5. Determination of carbohydrate content and productivity
Carbohydrate content in microalgae was measured by phenol-
sulfuric acid method. After freeze drying, samples of approximately
10 mg were blended with 72% H2SO4 and crushed with beaker in
mini-bead-beater (607EUR, BioSpec Products, USA), for 1 min each
time and repeated 7 times to ensure that the cell wall of microal-
gae was broken completely followed by water bath for 1 h at
30 C. Samples were diluted with distilled water to 4% sulfuric acid
and autoclaved at 121 C for 20 min and filtered using a 0.22 lm
filter. After diluted with distilled water at proper fold, 2 mL sample
was added with 1 mL 6% phenol and 5 ml sulfuric acid, and then
stabled for 30 min after which carbohydrate content was deter-
mined with UV spectrophotometer at a wavelength of 490 nm.
Carbohydrate content is calculated by the equation as follows:
Carbohydrate concentration mg=L  volume L
w %
Biomass mg
 100 3
Carbohydrate productivity is calculated by the equation as
follows:
Pcarbohydrate
Cumlative microalgae biomass prodctuion g  carbohydrate content mg=g
4
Working volume L  cultivation time d
Carbohydrate accumulation specific rate is calculated by:
d carbohydratecontent %
Q rate 5
d cultiv ationtime d
388 F. Tan et al. / Bioresource Technology 221 (2016) 385393

3. Results and discussion
3.1. Performance of five microalgae strains cultured in Modified BBM
Biomass accumulation, nutrients removal efficiency and carbo-
hydrate production of five microalgae strains cultured in Modified
BBM were invested. During 5-day cultivation, C. vulgaris ESP-6
grew most rapidly and achieved stable period firstly, accomplished
the largest biomass productivity of 757.59 mg/L/d, while C.
sorokiniana FACHB-275 reached the maximum biomass concentra-
tion of 3.16 g/L as well as the highest specific growth rate of
3.80 d1 (Table 2). It was easy to find that Chlorella species in this
study performed noticeably well for the maximum specific growth
rates were distinctly higher than that reported by related study
(specific growth rate ranging from 0.032 to 0.048 d1)
(Vidyashankar et al., 2015), showing a great potential on biomass
accumulation.
As for nitrate depletion, C. sorokiniana FACHB-275 surpassed
others by consuming nitrate both rapidly and completely within
43 h, achieving 98.4% nitrate removal efficiency, followed by S.
dimorphus FACHB-1266, C. vulgaris, Desmodesmus sp F51, and C.
vulgaris ESP-6 which had the nitrate removal efficiency of 95.0%
(48 h), 95.7% (48 h), 98.7% (60 h) and 95.4% (60 h), respectively.
These strains consumed up nitrate at intervals of less than a day,
suggesting that the microalgae strains were all in favor of this
abundant nutrition environment, leading small distinction made
on the time of nitrate depletion. It was precisely because that Mod-
ified BBM created favorable conditions for five strains and allowed
them to grow on its free will, the amount of phosphorus needed for
microalgae growth thus can be determined. Based on phosphorus
removal efficiency (Table 2), five strains required at least
23.2 mg/L phosphorus for five-day culture which provide compara-
ble data for further research conducted with biogas slurry.
Table 2 also illustrated carbohydrate content of five different
stains that cultured in Modified BBM. C. vulgaris ESP-6 obtained
the highest carbohydrate productivity of 364.34 mg/L/d and the
highest carbohydrate content of 50.99% which was higher than
the results reported by most of the related study (He et al., 2013;
Ji et al., 2015; Jiang et al., 2015), revealing a great potential on car-
bohydrate production.
3.2. Performance of five microalgae strains cultured in diluted biogas
slurry
The initial concentration of ammonia nitrogen in biogas slurry
was distinctly higher (10001200 mg/L) than that needed for
microalgae cultivation and high ammonia concentration restrained
growth. Meanwhile the concentration at which ammonia showed
certain inhibitory effect varied greatly with different algal strains
and culture conditions (Tam and Wong, 1996), five different strains
therefore were employed in 8-fold diluted biogas slurry to explore
their potential to survive in it and to combine microalgae cultiva-
tion with carbohydrates production and nutrients removal.
Delightfully, all the microalgae chosen in this study adapted to bio-
gas slurry easily and got to log phase rapidly with no inhibition
observed during the 5-day culture (Fig. 2a). Strain C. vulgaris
Esp6 surpassed the other four stains with the highest biomass pro-
ductivity of 643.60 mg/L/d, displaying enormous potential and
substantial competitive advantages. However, the biomass concen-
tration of C. sorokiniana FACHB-275, C. vulgaris, C. vulgaris ESP-6,
Desmodesmus sp F51 and S. dimorphus FACHB-1266 decreased
57.91%, 68.10%, 49.44%, 48.29% and 57.40% respectively in compar-
ison with those cultured in Modified BBM.
Table 3 presents carbohydrate contents of C. sorokiniana.
FACHB-275 (52.30 2.25%), C. vulgaris (54.82 2.52%), C. vulgaris
Esp6 (61.50 0.28%) and Desmodesmus sp F51 (51.85 1.66%) that

Table 2
Comparison of biomass, N, P removal and carbohydrate accumulation of five strains in Modified BBM.

Strains C. sorokiniana FACHB-275 C. vulgaris C. vulgaris ESP-6 Desmodesmus sp F51 S. dimorphus FACHB-1266
Biomass concentration (g/L) 3.16 0.06 2.79 0.08 2.69 0.15 2.92 0.08 2.23 0.15
Specific growth rate (d1) 3.80 0.17 2.68 0.06 1.34 0.05 2.28 0.21 1.53 0.04
Biomass productivity (mg/L/d) max 755.10 19.15 649.10 8.27 757.59 61.86 736.26 0.24 737.77 53.85
Nitrate removal efficiency (%) 98.41 0.45 98.07 0.09 97.52 0.83 98.68 0.03 97.52 0.34
Nitrate removal rate(mg/L/d) 71.43 1.46 62.02 1.31 51.09 0.24 49.98 0.39 61.60 2.09
Phosphorus removal efficiency (%) 99.44 0.23 100.00 0.00 82.67 7.26 99.11 0.75 93.63 5.58
Phosphorus removal rate(mg/L/d) 7.31 0.38 10.55 0.20 6.40 0.79 5.96 0.51 6.27 1.00
Carbohydrate content (%) 49.97 1.46 40.64 3.46 50.99 5.28 31.18 2.61 45.22 4.76
Carbohydrate productivity(mg/L/d) 334.91 16.95 225.45 10.41 364.34 40.92 181.64 3.33 263.65 68.54

Table 3
Comparison of biomass accumulation, nitrogen removal and carbohydrate accumulation of five strains in diluted biogas slurry.

Component Initial value Final value

C. sorokiniana FACHB- C. vulgaris C. vulgaris ESP- Desmodesmus sp S. dimorphus. FACHB-
275 6 F51 1266
TN 146.64 8.12 73.26 4.66 49.85 4.13 108.35 5.50 71.26 0.77 52.25 3.42
NH4-N 140.03 3.49 62.03 4.59 26.49 3.31 100.00 2.90 60.25 0.42 56.89 2.52
TP 1.242.19 0.51 0.01 0.58 0.10 0.23 0.49 0.02 0.70 0.00
PO3
4 -P 2.01 0.04 0.09 0.01 0.09 0.00 0.17 0.01 0.12 0.00 0.08 0.01
TN removal efficiency (%) / 50.06 0.41 65.87 4.71 28.28 4.84 51.35 2.17 64.38 0.36
NH4-N removal efficiency (%) / 54.23 3.13 80.74 1.85 29.43 1.67 57.68 0.14 60.28 1.77
pH 7.91 7.41 6.76 7.81 7.08 6.83
Biomass concentration (g/L) / 1.33 0.14 0.89 0.10 1.36 0.22 1.51 0.02 0.95 0.09
Specific growth rate (d1) / 2.73 0.02 2.67 0.05 1.59 0.11 2.20 0.00 1.11 0.02
Biomass productivity (mg/L/d) max / 617.10 0.18 504.20 20.73 643.60 89.39 268.35 62.69 538.56 20.05
Carbohydrate (%)max / 52.30 2.25 54.82 2.52 61.50 0.28 51.85 1.66 39.20 1.61
Carbohydrate productivity (g/L/d) / 285.19 31.18 184.92 14.98 341.54 54.19 157.57 17.66 140.30 12.26

Note: all data are presented as mean SD.

 F. Tan et al. / Bioresource Technology 221 (2016) 385393 389

were higher than S. dimorphus. FACHB-1266 (39.20 1.61%). The cultured in this study having ammonia made up most of that
results clearly proved the potential of culturing these four strains 95.5% (Table 1). Fig. 2(b) and (d) indicated that the removal ten-
in biogas slurry for carbohydrate production. However, since the dency of TN and NH4-N were alike, suggesting nitrate and organic
carbohydrate content is not the only issue that matters when nitrogen played a very weak role in algae growth on biogas slurry,
comes to this study, the ammonia removal efficiency or rates also which also confirmed that vast majority nitrogen resource that
should be taken into consideration for biogas slurry treatment. supported the microalgae growth was ammonia nitrogen.
Nitrogen source consist of nitrate, ammonia, urea and other Table 3 shows the ammonia removal efficiency of C. sorokiniana
organic nitrogen, while biogas slurry at which algae strain were FACHB-275 (54.22%), C. vulgaris (80.71%), C. vulgaris Esp6 (29.43%),

3 160
C.sorokiniana. FACHB-275 (a) (b)
C. vulgaris 140
Biomass concentration (g/L)

C. vulgaris ESP-6
S.dimorphus FACHB-1266 120
2

NH4-N (mg/L)
Desmodesmus sp F51 100
80

1 60
40
20
0 0
0 1 2 3 4 5 0 1 2 3 4 5
180
2.0 (c) 160 (d)
140
1.5 120
P-PO3-4 (mg/L)

TN (mg/L)

100
1.0 80
60
0.5 40
20
0.0 0
0 1 2 3 4 5 0 1 2 3 4 5
Time (d) Time (d)

Fig. 2. Time-course record of (a) biomass concentration, (b) ammonia concentration, (c) phosphorus concentration and (d) total nitrogen concentration in diluted biogas
slurry.

5
Esp6-BS (a) Cs-BS (b) Cv-BS (c)
Esp6-BS+P Cs-BS+P Cv-BS+P
4 Esp6-BS+Mg Cs-BS+Mg Cv-BS+Mg
Biomass concentration (g/L)

Esp6-BS+P+Mg Cs-BS+P+Mg Cv-BS+P+Mg

3 Esp6-Modified BBM Cs-Modified BBM Cv-Modified BBM

0
180
160
Nitrogen concentration (mg/L)

140
120
100
80
60
40
20
0
0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6
Time (d) Time (d) Time (d)

Fig. 3. Time-course record of biomass concentration and nitrogen concentration of (a) C. vulgaris ESP-6, (b) C. sorokiniana FACHB-275 and (c) C. vulgaris in diluted biogas
slurry with nutrients supply and Modified Bold Basal 3N medium. aBS: Biogas Slurry, Modified BBM: Modified Bold Basal 3N medium. bNitrogen concentration means
ammonia concentration in biogas slurry and biogas slurry with nutrients supply and means total nitrogen in Modified Bold Basal 3N medium.
390 F. Tan et al. / Bioresource Technology 221 (2016) 385393

90

Carbohydrate accumulation specific rate (mg/mg/d) Carbohydrate accumulation specific rate (mg/mg/d) Carbohydrate accumulation specific rate (mg/mg/d)
80 BS BS+P (a) 40 BS (a)
Content of carbohydrate (%)

BS+Mg BS+P+Mg BS+P

70 BS+Mg
Modified BBM 30
60 BS+P+Mg
50 Modified BBM
20
40
30 10
20
10 0
0
1 2 3 4 5 6 0 1 2 3 4 5 6
Esp6-Time (d) Esp6-Time (d)
90 40
80 BS BS+P (b) BS (b)
Content of carbohydrate (%)

BS+Mg BS+P+Mg BS+P

70 30
Modified BBM BS+Mg
60 BS+P+Mg
50 20 Modified BBM

40
30 10
20
10 0
0
1 2 3 4 5 6 0 1 2 3 4 5 6
Cs-Time (d) Cs-Time (d)
90
BS BS+P (c) 40
80 BS (c)
Content of carbohydrate (%)

BS+Mg BS+P+Mg BS+P

70
Modified BBM 30 BS+Mg
60 BS+P+Mg
50 Modified BBM
20
40
30 10
20
10 0
0
1 2 3 4 5 6 0 1 2 3 4 5 6
Cv-Time (d) Cv-Time (d)
Fig. 4. Time-course record of carbohydrate content of (a) C. vulgaris ESP-6, (b) C. Fig. 5. Time-course record of carbohydrate accumulation specific rate of (a) C.
sorokiniana FACHB-275 and (c) C. vulgaris in diluted biogas slurry with nutrients vulgaris ESP-6, (b) C. sorokiniana FACHB-275 and (c) C. vulgaris in diluted biogas
supply and Modified Bold Basal 3N medium. aBS: Biogas Slurry, Modified BBM: slurry with nutrients supply and Modified Bold Basal 3N medium. aBS: Biogas
Modified Bold Basal 3N medium. Slurry, Modified BBM: Modified Bold Basal 3N medium.

3.3. Nutrients optimism of diluted biogas slurry

Desmodesmus sp F51 (57.69%) and S. dimorphus FACHB-1266
(60.28%), among which C. vulgaris stood out in comparison with
others for the highest ammonia removal efficiency of 80.7% and
yet failed to lower the concentration of ammonia nitrogen to emis-
sion standard (<15 mg/L) by the end of the cultivation neither.
Given the high light illumination, suitable temperature and contin-
uously provided CO2, it was quite natural to believe that the nutri-
ents insufficiency except for nitrogen in the diluted biogas slurry
restrained the ammonia removal efficiency and biomass concen-
tration. Further research must be conducted in the aspect of nutri-
ents optimism in biogas slurry with C. vulgaris Esp6 and C.
sorokiniana FACHB-275 which were selected for high carbohydrate
productivity while C. vulgaris was selected for high ammonia
removal efficiency.
In order to combine carbohydrate production with biogas
slurry treatment by microalgae, ammonia, one of the main pollu-
tants for water eutrophication, has to be declined to emission
standard and thus extra nutrients were desperately needed
based on previous research. Table 1 showed the initial phospho-
rus concentration in 8-fold diluted biogas slurry is 2.18 mg/L
which was far less than sufficient when compared to phosphorus
content in Modified BBM. According to Redfield ratio, microalgae
have an elemental ratio of nitrogen to phosphorus of approxi-
mately 16:1 (Baird and Middleton, 2004). Hillebrand also points
out that while N:P ratios exceed 22 indicated P limitation
(Hillebrand and Sommer, 1999). However, N:P ratios of 73:1
(Table 1) was presented in this study. Fig. 2(C) indicated that
 F. Tan et al. / Bioresource Technology 221 (2016) 385393 391

phosphorus was significantly low at the beginning and was
almost consumed up within a day, leaving a severe living envi-
ronment, resulting in adversely impact on microalgae growth.
It seems that phosphorus deficiency was one of the main reasons
why the biomass concentration of all microalgae was lower that
cultured in Modified BBM.
After preliminary experiment with C. vulgaris ESP-6 (Fig. S1), it
was able to determine that both phosphorus and magnesium were
necessary for ammonia removal and biomass accumulation, while
nutrients like Na and other minor elements were not. Therefore,
modified biogas slurry with phosphorus and magnesium added
was employed to cultivate 3 strains.
3.4. Biomass, ammonia and phosphorus concentration with time of 3
strains cultured in diluted biogas slurry with phosphorus and
magnesium added
accompanied by a form of energy input (Cai et al., 2013). On the
other hand, magnesium consumption contributes to biomass pro-
duction and is also necessary for chlorophyII production (Sydney
et al., 2010). With more chlorophyII produced after magnesium
added to waste water, the efficiency of photosynthesis hence
improved greatly. Therefore, phosphorus and magnesium both
affected the efficiency of metabolism and photosynthesis, increas-
ing cell density, ammonia removal efficiency and rates as the
results.
Therefore, C. vulgaris Esp-6 and C. sorokiniana FACHB-275 were
considered to culture in biogas slurry with phosphorus and magne-
sium added while C. vulgaris needs only phosphorus as nutrients
supply when cultured in biogas slurry to achieve higher cell den-
sity, ammonia removal efficiency and removal rates.

The results showed in Fig. 3 met the expectation that both bio- 0.6
BS BS+P (a)

Carbohydrate productivity (mg/L/d)

mass concentration and removal efficiency were improved greatly
0.5 BS+Mg BS+P+Mg
with phosphorus and magnesium added in biogas slurry. Culturing
Modified BBM
strains in biogas slurry without nutrients supply resulted in poor 0.4
growth and low ammonia removal efficiency. While phosphorus
was added into diluted biogas slurry, cell density or ammonia 0.3
removal efficiency of stains C. vulgaris Esp-6 and C. sorokiniana
FACHB-275 were barely improved, approaching 44.56% and 0.2
66.17% removal efficiency respectively. But the effect of phospho-
rus was quite different when added into biogas slurry of C. vulgaris 0.1
which grew fast and consumed up ammonia rapidly within
1.5 days. Biomass concentration and ammonia removal rates of C. 0.0
vulgaris were significantly higher than that cultured in biogas
0 1 2 3 4 5 6
slurry without phosphorus added with an increase of 234.48% Esp6-Time (d)
and 345.18% respectively.
0.6
With magnesium added into diluted biogas slurry, biomass con-
BS BS+P (b)
Carbohydrate productivity (mg/L/d)

centration of C. vulgaris Esp-6, C. sorokiniana FACHB-275 and C. vul- 0.5 BS+Mg BS+P+Mg
garis improved 18.12%, 42.33%, and 101.15% compared with those Modified BBM
cultured in biogas slurry without phosphorus and magnesium 0.4
added, while ammonia removal efficiency increased greatly and
reached 95.72%, 93.92% and 97.61% by the end of the cultivation. 0.3
Nevertheless, biomass concentration and ammonia removal rate
were still not yet optimal when compared to those cultured in 0.2
Modified BBM, therefore magnesium and phosphorus were added
into biogas slurry simultaneously. 0.1
With phosphorus and magnesium presented in biogas slurry, C.
0.0
vulgaris Esp-6, C. sorokiniana FACHB-275 and C. vulgaris obtained
the maximum biomass concentration of 2.49 g/L, 3.46 g/L and 0 1 2 3 4 5 6
2.93 g/L, which is 67.11%, 152.55% and 236.78% higher than that Cs-Time (d)
cultured in biogas slurry without nutrients supplement and were 0.6
even comparable to that cultured in Modified BBM, lowered the BS BS+P (c)
Carbohydrate productivity (mg/L/d)

ammonia content to emission standard and reached ammonia 0.5 BS+Mg BS+P+Mg
removal efficiency of 96.26%, 97.58% and 98.79%. Meanwhile, C. Modified BBM
vulgaris Esp-6, C. sorokiniana FACHB-275 and C. vulgaris had not 0.4
only higher removal efficiency but also faster removal rate. Three
strains all depleted approximately 130 mg/L ammonia within 0.3
2 days, approaching ammonia removal rates of 91.68 mg/L/d,
87.10 mg/L/d and 76.11 mg/L/d which were at least tripled in gen- 0.2
eral than that cultured in biogas slurry without phosphorus and
0.1
magnesium added. In addition, C. vulgaris Esp-6, C. sorokiniana
FACHB-275 and C. vulgaris achieved phosphorus removal efficiency
0.0
of more than 97.60% (Fig. S2).
Phosphorus and magnesium had such a significant impact on 0 1 2 3 4 5 6
the improvement of biomass and ammonia removal can be traced Cv-Time (d)
to the role they both played in microalgae metabolism and photo-
Fig. 6. Time-course record of carbohydrate productivity of (a) C. vulgaris ESP-6, (b)
synthesis. During microalgae metabolism, phosphorus, involves C. sorokiniana FACHB-275 and (c) C. vulgaris in diluted biogas slurry with nutrients
the generation of ATP from adenosine diphosphate (ADP) by incor- supply and Modified Bold Basal 3N medium. aBS: Biogas Slurry, Modified BBM:
porated into organic compounds through phosphorylation, which Modified Bold Basal 3N medium.
392 F. Tan et al. / Bioresource Technology 221 (2016) 385393

3.5. Time-course profiles of carbohydrate production of 3 strains
Nutrients supplement including phosphorus and magnesium in
biogas slurry had impact on biomass concentration, ammonia
utilization as well as carbohydrate production of three strains
(Fig. 4). When cultured in Modified BBM, three stains accumulated
carbohydrate gradually in first two days after which carbohydrate
content rose largely while ammonia came to depletion in day 3.
Carbohydrate content increased greatly after external nitrogen
exhausted had been commonly realized and employed as nitrogen
starvation strategy to accumulate carbohydrate (Ho et al., 2013).
Culturing microalgae strains in biogas slurry without nutrients
supplement was utterly different in carbohydrate accumulation for
all strains (including Desmodesmus sp F51 and S. dimorphus FACHB-
1266) which improved highest carbohydrate content by 526.2%
than that cultured in Modified BBM and accumulated large quan-
tity of carbohydrate since the beginning of cultivation. This result
stood out particularly when made comparison between those cul-
tured in biogas slurry and Modified BBM in first three days. Taking
day 1 for example, the carbohydrate content of C. vulgaris ESP-6
was 39.00 1.60% which was 1.354.79 times higher than that
cultured under other four conditions. The carbohydrate content
of C. sorokiniana FACHB-275 and C. vulgaris were 24.66 10.23%
and 34.23 4.05% respectively in day 1, which were
0.9719.55 times and 1.4019.37 times higher than results came
from the other four conditions. Fig. 5 verified this particularity of
using diluted biogas slurry as medium for microalgae to produce
carbohydrate and convincingly showed that all three strains had
distinctly higher carbohydrate accumulation specific rate in first
three days. In other words, phosphorus and magnesium insuffi-
cient biogas slurry created stressing environment for microalgae,
resulting in earlier timing of carbohydrate accumulation and mak-
ing biogas slurry the more suitable medium for improvement of
carbohydrate content, which was proposed for the first time in
all related studies.
After phosphorus or magnesium added into biogas slurry sepa-
rately as nutrients supply, both biomass concentration and ammo-
nia removal efficiency were effected greatly and the pattern of
carbohydrate accumulation of three microalgae strains also was
altered based on Figs. 4 and 5. Three stains no longer exhibited
high carbohydrate content in the first day, with the highest carbo-
hydrate content decreased after 6 days cultivation. Meanwhile,
with phosphorus and magnesium added into biogas slurry simulta-
neously, the tendency of carbohydrate accumulation was more
similar to that cultured in Modified BBM, starting from low content
in first day or first two days then rising up after nitrogen con-
sumed. Therefore, the results proved the absence of phosphorus
and magnesium that can be adverse for biomass accumulation,
played an important role in carbohydrate production. Magnesium
was recognized and proved as the influence factor for carbohydrate
accumulation for the first time.
Fig. 6 showed carbohydrate productivity variation with time of
three strains that all obtained the highest carbohydrate productiv-
ity in biogas slurry with phosphorus and magnesium added,
among which C. vulgaris Esp-6 attained the highest carbohydrate
productivity of 388.34 29.54 mg/L/d (Table 4), which was much
higher than that reported by relative studies (He et al., 2013; Ji
et al., 2015, 2014). Meanwhile, it was worth noticing that when
cultured C. vulgaris Esp-6 and C. sorokiniana FACHB-275 in biogas
slurry, biogas slurry with phosphorus added and biogas slurry with
magnesium added, carbohydrate productivity of these two strains
peaked in day 1 or day 2, while cultured in biogas slurry with both
phosphorus and magnesium added and Modified BBM, C. vulgaris
Esp-6 and C. sorokiniana FACHB-275 delayed the stage that the
highest carbohydrate productivity appeared, which showed in
day 3 and day 4 respectively. The distinction of these two group
(BS, BS with phosphorus and BS with magnesium vs BS with phos-
phorus and magnesium, Modified BBM) can be explained by mak-
ing connection between microalgae growth and carbohydrate
production. It was reasonable to assume that when phosphorus
and magnesium were sufficient, microalgae utilized ammonia for
cell growth with biomass concentration increase at first, then accu-
mulated carbohydrate after nitrogen exhaustion. But if there was
deficiency in phosphorus or magnesium content which considered
to a stress environment, microalgae strains accumulated carbohy-
drate since the beginning, resulting the earlier timing when carbo-
hydrate productivity peaked, particularly when phosphorus and
magnesium were severely insufficient concurrently, microalgae
strains urgently rose a great quantity of carbohydrate in the very
beginning. This conclusion based on C. vulgaris Esp-6 and C.
sorokiniana FACHB-275 also applies to C. vulgaris which could grow
normally after phosphorus added into biogas slurry and had the
same growth curve as that when phosphorus and magnesium both
added. It corresponded to previous conclusion that phosphorus or

Table 4
Comparison of microalgae in wastewater.

Strains Light intensity Medium resource Ammonia Ammonia Carbohydrate Carbohydrate Reference
removal removal rate(mg/ (%)max productivity
efficiency (%) L/d) (mg/L/d)
Spirulina subsalsa 60 lmol m2 s1 Monosodium glutamate 98.8 15.24 17.69 48.98 Jiang et al. (2015)
factory
Scenedesmus 120 lmol Food wastewater N.D. N.D. 24.5 14.7 Ji et al. (2015)
obliquus photon m2 s1 treatment plant
Scenedesmus sp. 80 lmol m2 s1 Industrial plant 94 19.0 56.3 N.D. Jebali et al. (2015)
Chlorella vulgaris 600 lmol m2 s1 Biogas slurry 98.79 76.11 54.00 314.22 This research
Chlorella vulgaris 5500 lux Wastewater treatment 97 21.16 52.6 N.D. Gonzlez-
plant Fernndez et al.
(2016)
Chlorella. 7580 lmol m2 s1 Anaerobically digested 26 N.D. 26.2 6.9 N.D. Singh et al. (2011)
minutissima poultry litter effluent
Chlorella vulgaris 200 W/m2 Swine wastewater 39.6 2025 58.3 170 2 Wang et al. (2015)
JSC-6
Chlorella. 600 lmol m 2
s 1
Biogas slurry 97.58 87.10 48.38 322.61 This research
sorokiniana
FACHB-275
Chlorella. vulgaris 800010,000 lux Municipal wastewater N.D. N.D. 45 N.D. He et al. (2013)
ESP-6 treatment plant
Chlorella. vulgaris 600 lmol m2 s1 Biogas slurry 96.26 91.68 61.50 0.28 388.34 29.54 This research
ESP-6

N.D.: not determined.

 F. Tan et al. / Bioresource Technology 221 (2016) 385393
magnesium insufficient biogas slurry created stressing
environment for microalgae, resulting in earlier timing of carbohy-
drate accumulation.
Surprisingly, after cultured three different microalgae strains in
five different media, it was worth noticing that higher carbohy-
drate content was achieved in biogas slurry without nitrogen-
depletion the whole experimental time, suggesting that the
nitrogen starvation strategy was not the only way to increase car-
bohydrate content. Cultivate microalgae stains in biogas slurry
used in this study could result in higher carbohydrate content in
cell. Moreover, with significant improvement on carbohydrate
accumulation rate, the research may provide a new strategy to
accumulate carbohydrate. However, with high carbohydrate con-
tent and ammonia removal rate as two goals when coupling biogas
slurry treatment with carbohydrate production, phosphorus and
magnesium were required for C. vulgaris Esp-6 and C. sorokiniana
FACHB-275 while phosphorus was essential for C. vulgaris.
4. Conclusions
This research demonstrated the feasibility of utilizing diluted
biogas slurry for carbohydrate production with five promising
microalgae strains. Compared with Modified BBM, biogas slurry
attributed to higher carbohydrate content of five microalgae
among which C. vulgaris ESP-6 obtained the highest carbohydrate
content of 61.50% in diluted biogas slurry and the highest carbohy-
drate productivity of 388.34 mg/L/d in diluted biogas slurry with
phosphorus and magnesium added. Additionally, the absence of
phosphorus and magnesium that can be adverse for biomass accu-
mulation resulted in earlier timing of carbohydrate accumulation
and magnesium was firstly recognized and proved as the influence
factor for carbohydrate accumulation.
Acknowledgements
This work was supported by the National Basic Research
Program of China (2013CB733505), the National Natural Science
Foundation of China (41276101), the Fundamental Research Funds
for the Central Universities (20720160083) and the Science and
Technology Program of Xiamen, China (3502Z20143008).
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.2016.09.
030.
References
APHA, 1998. Standard Methods for the Examination of Water and Wastewater.
American Public Health Association, Washington, DC, USA.
393
Baird, M.E., Middleton, J.H., 2004. On relating physical limits to the carbon: nitrogen
ratio of unicellular algae and benthic plants. J. Mar. Syst. 49 (14), 169175.
Cai, T., Park, S.Y., Li, Y., 2013. Nutrient recovery from wastewater streams by
microalgae: status and prospects. Renewable Sustainable Energy Rev. 19, 360
369.
Gonzlez-Fernndez, C., Mahdy, A., Ballesteros, I., Ballesteros, M., 2016. Impact of
temperature and photoperiod on anaerobic biodegradability of microalgae
grown in urban wastewater. Int. Biodeterior. Biodegrad. 106, 1623.
He, P.J., Mao, B., Shen, C.M., Shao, L.M., Lee, D.J., Chang, J.S., 2013. Cultivation of
Chlorella vulgaris on wastewater containing high levels of ammonia for
biodiesel production. Bioresour. Technol. 129, 177181.
Hillebrand, H.S., Sommer, U., 1999. The nutrient stoichiometry of benthic microalgal
growth: redfield proportions are optimal. Limnol. Oceanogr. 44, 440446.
Ho, S.H., Huang, S.W., Chen, C.Y., Hasunuma, T., Kondo, A., Chang, J.S., 2013.
Characterization and optimization of carbohydrate production from an
indigenous microalga Chlorella vulgaris FSP-E. Bioresour. Technol. 135, 157
165.
Huang, H.Y., Cao, J.L., Wu, H.S., Ye, X.M., Ma, Y., Yu, J.G., Shen, Q.R., Chang, Z.Z., 2014.
Elevated methane emissions from a paddy field in southeast China occur after
applying anaerobic digestion slurry. GCB Bioenergy 6 (5), 465472.
Jebali, A., Acien, F.G., Gomez, C., Fernandez-Sevilla, J.M., Mhiri, N., Karray, F., Dhouib,
A., Molina-Grima, E., Sayadi, S., 2015. Selection of native Tunisian microalgae for
simultaneous wastewater treatment and biofuel production. Bioresour.
Technol. 198, 424430.
Ji, Y., Hu, W., Li, X., Ma, G., Song, M., Pei, H., 2014. Mixotrophic growth and
biochemical analysis of Chlorella vulgaris cultivated with diluted monosodium
glutamate wastewater. Bioresour. Technol. 152, 471476.
Ji, M.K., Yun, H.S., Park, S., Lee, H., Park, Y.T., Bae, S., Ham, J., Choi, J., 2015. Effect of
food wastewater on biomass production by a green microalga Scenedesmus
obliquus for bioenergy generation. Bioresour. Technol. 179, 624628.
Jiang, L., Pei, H., Hu, W., Ji, Y., Han, L., Ma, G., 2015. The feasibility of using complex
wastewater from a monosodium glutamate factory to cultivate Spirulina
subsalsa and accumulate biochemical composition. Bioresour. Technol. 180,
304310.
Kim, S., Lee, Y., Hwang, S.-J., 2013. Removal of nitrogen and phosphorus by Chlorella
sorokiniana cultured heterotrophically in ammonia and nitrate. Int. Biodeterior.
Biodegrad. 85, 511516.
Lam, M.K., Lee, K.T., 2012. Microalgae biofuels: a critical review of issues, problems
and the way forward. Biotechnol. Adv. 30 (3), 673690.
Maity, J.P., Bundschuh, J., Chen, C.-Y., Bhattacharya, P., 2014. Microalgae for third
generation biofuel production, mitigation of greenhouse gas emissions and
wastewater treatment: present and future perspectives A mini review. Energy
78, 104113.
Markou, G., Depraetere, O., Vandamme, D., Muylaert, K., 2015. Cultivation of
Chlorella vulgaris and Arthrospira platensis with recovered phosphorus from
wastewater by means of zeolite sorption. Int. J. Mol. Sci. 16 (2), 42504264.
Quiroz Arita, C.E., Peebles, C., Bradley, T.H., 2015. Scalability of combining
microalgae-based biofuels with wastewater facilities: a review. Algal Res. 9,
160169.
Singh, M., Reynolds, D.L., Das, K.C., 2011. Microalgal system for treatment of effluent
from poultry litter anaerobic digestion. Bioresour. Technol. 102 (23), 10841
10848.
Sydney, E.B., Sturm, W., de Carvalho, J.C., Thomaz-Soccol, V., Larroche, C., Pandey, A.,
Soccol, C.R., 2010. Potential carbon dioxide fixation by industrially important
microalgae. Bioresour. Technol. 101 (15), 58925896.
Tam, N.F.Y., Wong, Y.S., 1996. Effect of ammonia concentrations on growth of
Chlorella vulgaris and nitrogen removal from media. Bioresour. Technol. 57 (1),
4550.
Vidyashankar, S., VenuGopal, K.S., Swarnalatha, G.V., Kavitha, M.D., Chauhan, V.S.,
Ravi, R., Bansal, A.K., Singh, R., Pande, A., Ravishankar, G.A., Sarada, R., 2015.
Characterization of fatty acids and hydrocarbons of chlorophycean microalgae
towards their use as biofuel source. Biomass Bioenergy 77, 7591.
Wang, Y., Guo, W., Yen, H.W., Ho, S.H., Lo, Y.C., Cheng, C.L., Ren, N., Chang, J.S., 2015.
Cultivation of Chlorella vulgaris JSC-6 with swine wastewater for simultaneous
nutrient/COD removal and carbohydrate production. Bioresour. Technol. 198,
619625.