Bioresource Technology 242 (2017) 714

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

Nutrients and COD removal of swine wastewater with an isolated

microalgal strain Neochloris aquatica CL-M1 accumulating high
carbohydrate content used for biobutanol production
Yue Wang a,b, Shih-Hsin Ho c, Chieh-Lun Cheng d, Dillirani Nagarajan d, Wan-Qian Guo c, Chiayi Lin d,
Shuangfei Li a, Nanqi Ren c, Jo-Shu Chang c,d,e,
a
Shenzhen Key Laboratory of Marine Bioresources and Ecology, Shenzhen University, Shenzhen 518060, China
b
Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University,
Shenzhen 518060, China
c
State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin, China
d
Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan
e
Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan 701, Taiwan

h i g h l i g h t s

Carbohydrate-rich microalga Neochloris aquatica CL-M1 was grown in swine wastewater.

Effect of temp., light and N/P ratio on nutrient removal/cell growth was studied.

81.7% COD removal and 96.2% NH3-N removal was achieved with the microalgal growth.
1

Biomass conc. and carbohydrate content reached 6.10 g L and 50.46%, respectively.

The pretreated algal biomass was used to produce butanol with good efficiency.

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

Article history: In this study, a carbohydrate-rich microalga Neochloris aquatica CL-M1 was adapted to grow in swine
Received 25 January 2017 wastewater. The effects of cultivation conditions (i.e., temperature, light intensity or N/P ratio) on COD/
Received in revised form 20 March 2017 nutrients removal and carbohydrate-rich biomass production were investigated. The results indicate that
Accepted 21 March 2017
the highest COD removal (81.7%) and NH3-N removal (96.2%) was achieved at 150 mmol m2 s1 light
Available online 24 March 2017
intensity, 25 C and N/P ratio = 1.5/1. The highest biomass concentration and carbohydrate content was
6.10 g L1 and 50.46%, respectively, when N/P ratio = 5/1. The resulting carbohydrate-rich microalgal bio-
Keywords:
mass was pretreated and used as a feedstock for butanol fermentation. With the initial sugar concentra-
Swine wastewater
Neochloris aquatica CL-M1
tion of 48.7 g L1 glucose and 3.4 g L1 xylose in the pretreated biomass, the butanol concentration, yield,
Microalgae and productivity were 12.0 g L1, 0.60 mol mol1 sugar, and 0.89 g L1 h1, respectively, indicating the
Carbohydrate high potential of using Neochloris aquatica CL-M1 for butanol fermentation.
COD/nutrient removal 2017 Elsevier Ltd. All rights reserved.
Butanol fermentation

1. Introduction
Global energy consumption is projected to increase, the carbon
intensity (the amount of carbon emitted in weight, per unit of
energy supplied) in 2050 will reach approximately 0.45 kg of C
yr1 W1 according to the Intergovernmental Panel on Climate
Change projection (Intergovernmental Panel on Climate Change
IPCC). Therefore, the only environmentally friendly way for solving
global energy demand is through a significant contribution of solar
Corresponding author at: Department of Chemical Engineering, National Cheng
Kung University, Tainan, Taiwan.
E-mail address: changjs@mail.ncku.edu.tw (J.-S. Chang).
energy and gas carbon (CO2), which is present in excess in our
atmosphere, to the total energy mix (Huntley and Redalje, 2006).
Photosynthetic plants are well equipped to harness both these
energy resources, as they can fix gaseous carbon using sunlight
as energy source, but they have very low photosynthetic efficiency.
On the other hand, microscopic photosynthetic organisms like
microalgae have much higher photosynthetic efficiency compared
to terrestrial plants (Huntley and Redalje, 2006). Also, microalgae
have the added advantage of very short life cycles, very simple
nutritional requirements and the ability to grow on marginal land.
Thus, microalgae can be cultivated as an alternative feedstock for
biofuel production. Biofuel production with microalgal biomass

http://dx.doi.org/10.1016/j.biortech.2017.03.122
0960-8524/ 2017 Elsevier Ltd. All rights reserved.
8 Y. Wang et al. / Bioresource Technology 242 (2017) 714
can be based on the two energy dense components of microalgae:
transesterification of microalgal lipids for biodiesel production
(Mandotra et al., 2016) and fermentation of microalgal carbohy-
drates for bioethanol and biobutanol production (Ho et al., 2012;
Wang et al., 2016). Both biodiesel production and fermentation
are dependent on microalgal biomass cultivation and concomitant
accumulation of lipids/carbohydrates. However, cultivation of
microalgae are limited by several issues, such as high medium cost
(Slade and Bauen, 2013), high contamination risk in case of out-
door cultivation which is the most preferred method economically
(Peng et al., 2015), and the extensive energy input for large-scale
microalgae cultivation (Breuer et al., 2013). Several factors, includ-
ing light intensity, temperature, and nutrients composition and
concentration, could affect microalgae growth. Light supply is
one of the most important factors to be considered for microalgal
growth (Yeh et al., 2010). In general, microalgae cultivation can
occur in variable light intensities: complete darkness, light limita-
tion, light saturation and light inhibition. Temperature influence on
microalgal growth has also been modeled in various ways (Ruiz-
Martinez et al., 2015), and treated as a crucial factor for outdoor
microalgae growth (Ras et al., 2013).
Cultivation of microalgae with wastewater is considered as an
ideal low-cost scenario for obtaining microalgal biomass as a feed-
stock for biofuel use. Microalgae cultivation with wastewater has
been intensively studied recently, with a focus on the production
of algal biomass and the associated removal of inorganic nutrients
from a wide variety of wastewaters (Aravantinou et al., 2013; Cho
et al., 2011; Wu et al., 2012). Swine wastewater is the effluent from
the swine farms and the high nitrogen content makes it a suitable
source of nutrients for the cultivation of microalgae (Wang et al.,
2015). In previous studies, the most investigated microalga for
swine wastewater treatment and biomass production was from
the genus Chlorella (Godos et al., 2009; Kao et al., 2012; Wang
et al., 2010). In our earlier study, we used Chlorella vulgaris JSC-6
for treatment of swine wastewater and carbohydrate-rich biomass
production (Wang et al., 2015). It was suggested that cultivating
microalgal biomass for lipid production in nutrient rich wastewa-
ter is not a reasonable approach, as lipid accumulation is often trig-
gered by nutrient limitation (Wang et al., 2015). Carbohydrate
accumulation, on the other hand, can be achieved under optimal
N/P ratios. The feasibility of using Neochloris sp. for lipid-rich bio-
mass production and nutrients removal in different kinds of
wastewaters has been investigated, such as municipal wastewater
(Wang and Lan, 2011), agricultural wastewater (Chinnasamy et al.,
2010), industrial wastewater (Wu et al., 2012), and anaerobic
digestates of vinasse (Olgun et al., 2015). Very few reports are
available on the use of Neochloris sp. for the treatment of swine
wastewater and biomass production. Moreover, Neochloris species,
mainly Neochloris oleoabundans has been employed for biodiesel
production because of the promisingly high level intracellular lipid
accumulation (Olgun et al., 2015; Yoon et al., 2015). However, few
reports explored the ability of Neochloris species for carbohydrate
production and wastewater treatment (Olgun et al., 2015).
In this study, a carbohydrate-rich microalga Neochloris aquatica
CL-M1 was used to simultaneously remove nutrients and COD
from swine wastewater. The harvested carbohydrate-rich microal-
gal biomass was used as a carbon source for further conversion to
value-added products through fermentation.
2. Material and methods
2.1. Microalgal strain and pre-culture conditions
The microalga used in this study is Neochloris aquatica CL-M1,
isolated from fresh water in Southern Taiwan. BG-11 medium
was used as pre-culture medium with continuously supplied
carbon dioxide (2.5%) at a flow rate of 0.2 vvm (volume/working
volume/min), 25 2 C temperature and a light intensity of
150 mmol m2 s1.
2.2. Cultivation of Neochloris aquatica CL-M1under different
conditions
The swine wastewater used in the experiments was collected
from a pig farm owned by Taiwan Sugar Company in Southern Tai-
wan. The original swine wastewater was first centrifuged at
9000 rpm for 5 min to remove insoluble solids before being auto-
claved. The COD, NO3-N, NH3-N and PO4-P in original swine
wastewater used in this study were 2070 71 mg L1,
15.3 6.5 mg L1, 220.6 12.9 mg L1 and 12.7 2.2 mg L1,
respectively. The original swine wastewater had a high pH value
of around 9.1, and the pH was adjusted to approximately 7.5 with
5 N HCl. The wastewater was directly used for the cultivation of
microalgae without any dilution or modification. There was no
pH control during the cultivation of microalgae. The microalga
was cultivated in a 200-mL glass photobioreactor illuminated by
external light sources (i.e., white fluorescent lamps). All experi-
ments were conducted in batch mode and the experiment was ter-
minated after the system reached steady state (No decrease in pH,
growth curve in decline phase and no decrease in nutrient level).
The microalgae growth, wastewater treatment, and carbohydrate
accumulation were compared at different light intensities
(50200 mmol m2 s1), different temperature (1540 C), and
N/P ratios (1.5:115:1).
2.3. Fermentative bacterial strain and its growth condition
Clostridium acetobutylicum ATCC824 was used for butanol
fermentation. The growth medium of C. acetobutylicum ATCC824
consisted of (g L1): glucose, 60; tryptone, 1; yeast extract, 5;
p-aminobenzoic acid, 0.01; Na2SO4, 0.18; KH2PO4, 0.175; and Bio-
tin, 0.01. The initial pH was adjusted to 4.8. The butanol fermenta-
tion was conducted at a 37 C without pH control.
2.4. Hydrolysis of Neochloris aquatica CL-M1 biomass for butanol
fermentation
The harvested and lyophilized Neochloris aquatica CL-M1 bio-
mass (about 10.98 g, sugar content 50%) was first pretreated with
1% NaOH for proteinous inhibitor removal (Wang et al., 2016).
The residual biomass was washed three times with water and
hydrolyzed with 3% H2SO4 for 20 min at 121 C. The acidic pH of
the hydrolysate was neutralized with CaCO3 and the precipitate
was removed by centrifugation. The clear supernatant thus
obtained was added to the medium for butanol fermentation.
2.5. Analytical methods
A volume of 5 mL microalgae suspension was collected every
day from each photobioreactor for nutrient analysis. The samples
were centrifuged at 10,000 rpm for 5 min. The collected super-
natant was then filtered through 0.45 mm cellulose membrane.
The filtrates were analyzed for COD (Potassium chromate method),
ammonia nitrogen (Nash-reagent spectrophotometric method),
nitrate nitrogen (phenol disulfonic acid method), and phosphorus
(molybdenum-antimony anti-spectrophotometric method). Har-
vested microalgal biomass was lyophilized at 50 C for 48 h for
components analysis.
The carbohydrate content in microalgal biomass was analyzed
according to the modified Quantitative saccharification method
reported by National Renewable Energy Laboratory (NREL) in
2008. Monosaccharides and soluble metabolites produced during
 Y. Wang et al. / Bioresource Technology 242 (2017) 714
the ABE fermentation were detected with HPLC equipped RID
detector, and the column type was ICSep156ICE-COREGEL 87H3
(Transgenomic, USA). The mobile phase was 0.008 N H2SO4, with
a flow rate of 0.4 mL/min, the injection sample volume was
0.2 mL and the column temperature was controlled at 70 C. The
amount of gas products produced (mainly H2 andCO2) was mea-
sured with a gas meter. The gas composition was analyzed using
a gas chromatograph (Model 9800, China Chromatography, Taipei,
Taiwan) with a thermal conductivity detector.
Concentration of microalgae and bacteria cells were estimated
based on the optical density values measured at 680 nm (OD680)
and 600 nm (OD600) with a spectrophotometer (model U-2001,
Hitachi, Tokyo, Japan). One way analysis of variance (ANOVA)
and multiple comparisons with LSD test on the experimental data
were conducted using SPSS statistics 17.0.
3. Results and discussions
3.1. Mixotrophic cultivation of Neochloris aquatica CL-M1
Mixotrophic cultivation seems to the most efficient cultivation
type for higher biomass production and accumulation of storage
reserves like lipids and carbohydrates (Ho et al., 2012; Wang
et al., 2015). Previous studies indicate that Neochloris oleoabundans
is a mixotrophic microalga, and is capable of utilizing organic car-
bon present agro-industrial waste product (Olgun et al., 2015). To
demonstrate the ability of Neochloris aquatica CL-M1 to grow in
mixotrophic mode, the microalga was grown in basal medium with
2.5% CO2 aeration, using acetate as an added organic carbon source
and the results are shown in Fig. 1a. A biomass concentration of
6.05 0.25 g L1 was obtained. The biomass increased nearly 30
times in the cultivation period of seven days. The specific growth
rate and biomass productivity were as high as 0.461 d1 and
0.82 g L1 d1, respectively. Nitrogen source was consumed rapidly
in first six days, with a consumption rate of 2.17 g L1 d1 KNO3.
After three days of nitrogen exhaustion, the carbohydrate in
microalgal cells increased from 30.27% to 46.13%. This result
demonstrated the ability of biomass and carbohydrate production
of Neochloris aquatica CL-M1 under mixotrophic conditions.
3.2. Cultivation of Neochloris aquatic CL-M1 in swine wastewater
Neochloris aquatica CL-M1 was also cultivated in undiluted
swine wastewater to check for growth and biomass production
Fig. 1. Biomass and carbohydrate production in (a) control, Basal medium with
0.5 g L1 acetic acid; (b) swine wastewater and (c) swine wastewater amended with
150 mg L1 phosphate.
9
in mixotrophic mode. As shown in Fig. 1b, a biomass concentration
of about 4 g L1 was achieved with the carbohydrate content of
38%, which was significant lower compared to the growth and car-
bohydrate accumulation in basal medium. Upon closer observa-
tion, it was found that low concentrations of phosphate present
in the swine wastewater (around 13 mg L1) is a limiting factor
for biomass production and carbohydrate accumulation. Thus,
the swine wastewater was supplemented with 150 mg L1 potas-
sium dihydrogen phosphate as a phosphate source before microal-
gal cultivation, as shown in Fig. 1c. The growth and biomass
concentration obtained with added phosphate was similar to the
basal medium, with slightly higher carbohydrate accumulation
(Fig. 1c). This indicates that the phosphate concentrations present
in this particular wastewater is insufficient to support efficient
microalgal biomass production and carbohydrate accumulation.
Similar results were obtained when cultivating Neochloris
oleoabundans in artificial wastewater, biomass production
increased with increasing phosphate concentration (i.e., P-PO3 4 )
to 108 mg L1 for avoiding phosphate limitation (Wang and Lan,
2011).
3.3. The microalgae growth on swine wastewater at different
irradiance
High biomass productivity is required to enhance the economic
feasibility of microalgal biofuel production. Above, light intensity
has been noticed as a vital parameter which can strongly affect
the accumulation of microalgal biomass productivity, particularly
for phototrophic strains (Ho et al., 2012). In addition, it has been
reported that the carbohydrate accumulation in microalgae is asso-
ciated with efficient photosynthetic activity, which is in turn con-
trolled by efficient irradiation (Xue et al., 2011). Evidence suggests
that the expression level of rate limiting enzyme (ADP-glucose
pyrophosphorylase) in starch synthesis is highly correlated to pho-
tosynthetic efficiency, which is influenced by irradiance (Tuncel
and Okita, 2013). Thus, in order to achieve better cell growth with
higher carbohydrate productivity, Neochloris aquatica CL-M1 was
cultivated in undiluted swine wastewater under increasing
irradiance (50200 mmol m2 s1) and the results are shown in
Fig. 2. Biomass concentration, NH3-N removal, COD removal and
carbohydrate accumulation increased with increase in light
intensity until 150 mmol m2 s1 and then decreased slightly at
200 mmol m2 s1. The maximum COD removal efficiencies
increased from 44.2 1.9% to 66.5 2.0% according to the light
intensities from 50 to 150 mmol m2 s1. The ammonia removal
efficiencies also increased from 10.3 0.5% to 79.2 3.0%. The
NO3-N removal was nearly 100% within a short period of time after
cultivation (data not shown). The PO4-P removal efficiencies were
almost 100% in all light intensities. It suggests that the supplied
PO4-P concentration was very low, resulting in the dramatic drop
in pH after the PO4-P consumption which is harmful to cell growth
or carbohydrate accumulation. In addition, although the carbohy-
drate content increased from 22.0% to 31.1% when the light inten-
sities increased from 50 to 200 mmol m2 s1 (Fig. 2a), the
carbohydrate content was decreased (compared to Fig. 1c), maybe
resulting from phosphorus limitation. Phosphorus is a known
macronutrient for the growth of microorganisms, essential for
the synthesis of nucleic acids and phospholipids. It was already
shown that NH3-N removal efficiency in wastewater is dependent
on the phosphate concentration (Wang and Lan, 2011).
3.4. The microalgae growth on swine wastewater at different
temperatures
Optimum growth temperatures of microalgae vary with species,
and within the species they can also be affected by environmental
10 Y. Wang et al. / Bioresource Technology 242 (2017) 714

Fig. 2. Biomass production and nutrient removal of Neochloris aquatica CL-M1 at Fig. 3. Growth and nutrient removal of Neochloris aquatica CL-M1at different
different light intensities. temperatures.

growth conditions (Ras et al., 2013). Temperature is a crucial factor the influence of temperature on ammonium removal rate, the com-
for ammonia removal from wastewaters and ammonia is the main promise between operation costs and removal efficiency can be
nutrient need to be removed from swine wastewater. Previous achieved.
reports suggested that temperature had great effect on the ammo-
nia uptake of microalgae (Ruiz-Martinez et al., 2015). Neochloris
3.5. The microalgae growth on swine wastewater at different N/P
aquatica Cl-M1 was cultivated in undiluted swine wastewater in
ratios
various temperatures (15 C, 25 C, 37 C and 40 C) and the results
are indicated in Fig. 3. The maximum biomass concentration
To obtain microalgal biomass with high carbohydrate content,
(3.7 g L1) and maximum carbohydrate content (33.2%) was
the N/P ratios in the swine wastewater was altered by external
obtained at 25 C. Biomass concentration, carbohydrate accumula-
addition of KH2PO4. The original N/P ratio of the swine wastewater
tion, NH3-N removal decreased with an increase in temperature
used in this study is 15/1 under the optimal cultivation condition
beyond 30 C. Both NO3-N (data not shown) and PO4-P (Fig. 3)
of 25 C and 150 mmol m2 s1. The optimum N/P ratio for cultiva-
removal efficiencies were nearly 100%. Maximum removal efficien-
tion of freshwater algae is known to be between 6.8 and 10.0, even
cies and biomass concentration was obtained at a temperature of
though the algae might encounter very high N/P ratios in their nat-
25 C.
ural habitats (Wang et al., 2010). The PO4-P concentration after N/P
The effect of temperature on microalgal nutrients utilization
ratio adjustment was 12.68, 48.46, 112.78, 149.00 mg L1 when the
can be described by Ratkowsky model (Eq. (1)) (Ratkowsky et al.,
N/P ratio was 15/1, 5/1, 2/1 and 1.5/1, respectively. The biomass
1983).
concentration increased from 3.6 g L1 to 6.1 g L1, and total carbo-
p
lT b  T  T min  f1  expc  T  T max g 1 hydrate content increased from 31.46% to 50.46% when the N/P
ratio decreased from 15/1 to 5/1 (Fig. 5a). The optimal biomass
where b and c are parameters with no biological meaning obtained
by data fitting, m(T) denotes the nutrient removal rate (mg L1 d1),
Table 1
Tmin and Tmax are the minimum and maximum temperatures, The nutrients removal rate with Neochloris aquatica CL-M1at different cultivation
respectively, at which the nutrient removal rate is zero. The least- temperatures.
squares fitting method was applied to fit the obtained data of
Temperature m (COD) m (NH3-N) m (PO4-P)
time-course nutrient removal profile for the calculation of the max- (C) (mg L1 d1) (mg L1 d1) (mg L1 d1)
imum nutrient removal rate with respect to culture temperature
15 68.5 16.3 12.3
(m(T)) form the slope of the profiles (Table 1). The experimental data 25 219.2 34.4 57.5
were predicted with Ratkowsky model using SPSS 17.0, obtaining 37 570.5 53.0 58.1
the following parameter values (Table 2). The maximum ammo- 40 225.7 13.6 11.5
nium removal rate obtained is in agreement with our observations m (COD): COD removal rate.
(Fig. 4). Ratkowskys model also predicts an optimum temperature m (NH3-N): ammonia removal rate.
for nutrients removal at around 30 C (Fig. 4). Taking into account m (PO4-P): phosphate removal rate.
 Y. Wang et al. / Bioresource Technology 242 (2017) 714
Table 2
Dynamic fit of the temperature effect on nutrients removal rate by Neochloris aquatica CL-M1 using Ratkowsky model (Eq. (1)) (Ratkowsky et al., 1983).
Predict Tmin Tmax
Ammonium 8.79 52.10
Phosphorus 14.87 58.56
COD 11.30 57.94
Fig. 4. Prediction of nutrients removal at different temperatures by Ratkowsky
model.
and carbohydrate production were similar with the values
obtained in control group (mixotrophic cultivation in Basal
medium with 0.5 g L1 acetic acid) (Fig. 1). These results are indica-
tive that altering the ratio of nutrients is important for the
enhancement of microalgal growth, efficient ammonium removal
and carbohydrate accumulation. Moreover, increase in PO4-P con-
centration did not increase the microalgal biomass production and
carbohydrate content further. When the N/P ratio decreased from
15/1 to 1.5/1, the COD removal efficiency increased from
66.0 1.9% to 81.7 1.9%, and ammonium removal efficiency
increased from 80.4 1.0% to 96.2 2.2% (Fig. 5b). However, the
PO4-P utilization efficiency decreased with a decrease in the N/P
ratio. When the N/P ratio decreased from 15/1 to 1.5/1, the PO4-P
utilization decreased from 100% to 46.3 0.7%, (Fig. 5b). In contrast
to cultivation at N/P ratio 1.5/1, N/P ratio of 5/1 resulted in a
slightly lower COD and ammonium removal efficiency of
73.5 0.5% and 92.5 1.0%, respectively. The biomass production
and PO4-P removal efficiency obtained at N/P ratio of 5/1 were
6.10 g L1 and 85.6 0.3%, respectively, which is much higher than
those obtained at N/P ratio = 1.5/1. Therefore, considering biomass
production, carbohydrate content, and nutrient removal efficiency,
the optimum N/P ratio for cultivation of Neochloris aquatica CL-M1
on wastewater seems to be 5/1.
In this study, a higher biomass production and nutrient removal
efficiency was obtained when compared with those reported in
previous studies (Table 3). It suggested that Neochloris aquatica
CL-M1 is promising for carbohydrate-rich biomass production
and wastewater treatment. The carbohydrate produced during cul-
tivation of Neochloris aquatica in swine wastewater is predomi-
nantly glucose and very little quantities of xylose. It is well
known that glucose is the preferred carbon source by fermenting
microorganisms and the presence of other pentoses will reduce
fermentation efficiency as the utilization of pentoses is by different
pathways and their uptake is inhibited in the presence of glucose
11
b c R2
2.77 0.007 0.749
1.18 0.014 0.844
1.48 0.056 0.813
Fig. 5. Biomass production and nutrient removal of Neochloris aquatica CL-M1
under different N/P ratios.
(Wang et al., 2014). The carbohydrates produced by cultivating
Chlorella vulgaris JSC-6 in swine wastewater also produced a
similar profile of carbohydrates produced, with glucose being the
predominant monosaccharide obtained after hydrolysis (Wang
et al., 2015). The carbohydrate-rich biomass can serve as feedstock
for various fermentations, which can reduce the cost for feedstock
production.
The statistical analysis (ANOVA) of the data obtained was per-
formed by SPSS 17.0 (Tables 4 and 5). The significance values of
cultivation condition effect to the COD, NH3-N, PO4-P removal, bio-
mass production, carbohydrate accumulation were 0.127, 0.118,
0.037, 0.004, and 0.011, respectively. This result demonstrated
the statistical significance of temperature, light intensity, and N/P
ratio effecting the COD/nutrients removal, biomass production
and carbohydrate accumulation. The results of LSD test suggested
that N/P ratio was a critical factor for nutrient removal and
carbohydrate-rich biomass production.
3.6. Butanol fermentation with pretreated microalgal biomass
Butanol fermentation was carried out by Clostridium aceto-
buylicum ATCC824, with the microalgal biomass hydrolysate as
carbon source. The initial sugar concentrations in the medium
were 48.7 g L1 glucose and 3.4 g L1 xylose. As shown in Fig. 6,
12 Y. Wang et al. / Bioresource Technology 242 (2017) 714

Table 3
Comparison of the performance of microalgae-based wastewater treatment.

Wastewater Microalgae strain Biomass (g L1) COD removal (%) NH3-N removal (%) References
Agro-zootechnical digestate C. vulgaris CCAP 211/11b Negligible 99.9 Franchino et al. (2013)
Piggery wastewater C. pyrenoidosa 0.3 55.4 91.2 Wang et al. (2012)
Piggery wastewater C. zofingiensis 2.962 79.84 81.03(TN) Zhu et al. (2013)
Anaerobically treated piggery wastewater C. vulgaris CY5 4.25 Increased TN concentration <5 mg L1 Marjakangas et al. (2015)
Swine wastewater C. vulgaris JSC-6 3.96 70 91.3 Wang et al. (2015)
Anaerobic effluents Neochloris oleoabundans 0.63 Negligible 98 Olgun et al. (2015)
Anaerobically digested dairy manure Neochloris oleoabundans 0.125 Negligible 95 Levine et al. (2011)
Municipal wastewaters Neochloris oleoabundans 2.1 Negligible 99 Wang and Lan, (2011)
Anaerobically treated piggery wastewater Neochloris oleoabundans 0.59 98% Olgun et al. (2015)
Swine wastewater Neochloris aquatica CL-M1 6.1 81.7 96.2 This study

Table 4
The results of one-way ANOVA analysis on the COD/nutrients removal and production of biomass and carbohydrate by Neochloris aquatica CL-M1.

Sum of squares df Mean squares F value Significance (p value)

COD removal 1464.538 2 732.269 2.617 0.127
NH3-N removal 2725.821 2 1362.910 2.738 0.118
PO4-P removal 1823.527 2 911.763 4.861 0.037
Biomass production 13.664 2 6.832 10.704 0.004
Carbohydrate accumulation 688.219 2 344.110 7.838 0.011

Table 5
Multiple comparisons using LSD test for the results of ANOVA analysis on COD/nutrient removal and production of biomass and carbohydrate by Neochloris aquatica CL-M1.

Parameter (I) (J) Mean difference (IJ) Std. error Sig. (p value)
COD removal T L 10.72885 11.82735 0.388
N 26.87885 11.82735 0.049
L T 10.72885 11.82735 0.388
N 16.15000 11.82735 0.205
N T 26.87885 11.82735 0.049
L 16.15000 11.82735 0.205
NH3-N removal T L 8.45730 15.77537 0.605
N 26.89270 15.77537 0.122
L T 8.45730 15.77537 0.605
N 35.35000 15.77537 0.052
N T 26.89270 15.77537 0.122
L 35.35000 15.77537 0.052
PO4-P removal T L 0.00000 9.68392 1.000
N 26.15000 9.68392 0.024
L T 0.00000 9.68392 1.000
N 26.15000 9.68392 0.024
N T 26.15000 9.68392 0.024
L 26.15000 9.68392 0.024
Biomass T L 0.80000 0.56491 0.190
N 2.55500 0.56491 0.001
L T 0.80000 0.56491 0.190
N 1.75500 0.56491 0.013
N T 2.55500 0.56491 0.001
L 1.75500 0.56491 0.013
Carbohydrate accumulation T L 5.38230 4.68533 0.280
N 18.06500 4.68533 0.004
L T 5.38230 4.68533 0.280
N 12.68270 4.68533 0.004
N T 18.06500 4.68533 0.004
L 12.68270 4.68533 0.0224

T: temperature.
L: light.
N: N/P ratio.
Std. error: standard error.
Sig.: significance.

the total solvent production (i.e., butanol, acetone and ethanol)
reached a concentration of 19.61 g L1 with an A:B:E ratio of
2:7:1. The butanol concentration, butanol yield, and butanol pro-
ductivity were 12.00 g L1, 0.60 mol mol1 sugar (0.25 g g1 sugar),
and 0.89 g L1 h1, respectively. The efficiency of the conversion
process from substrate to butanol was 0.12 g g1 biomass. In addi-
tion, the rate of butanol formation over substrate for alkali-acid
treated biomass was 0.0085 g g1 substrate h1. This value was a
little higher than our previous reported data (Wang et al., 2016)
and most other relevant studies (Wang et al., 2014), suggesting
 Y. Wang et al. / Bioresource Technology 242 (2017) 714
Fig. 6. Butanol fermentation using the hydrolysate of the biomass of Neochloris
aquatica CL-M1. The microalgal hydrolysate was obtained by alkali pretreatment
followed by acid (H2SO4) hydrolysis.
that butanol production from microalgal biomass hydrolysate is
affected by the microalgal strain. The ABE fermentation was also
accompanied by production of 1478 mL L1 hydrogen during
acidogenesis phase. The hydrogen yield and productivity were
0.22 mol mol1 sugar and 68.8 mL L1 h1, respectively. In
addition, glucose consumption was nearly complete (99.9% glucose
and 88.4% xylose), indicating the absence of any inhibitors in the
hydrolysate.
4. Conclusion
In this study, light intensity, temperature and N/P ratio were
optimized for the cultivation of Neochloris aquatica CL-M1 using
swine wastewater. Under optimal conditions with a N/P ratio of
5/1, the CL-M1 strain can efficiently utilize nutrients and COD in
swine wastewater for cell growth and carbohydrate accumulation,
giving high COD (73.5%) and nitrogen (96.2%) removal efficiencies
as well as high biomass concentration (6.10 g L1) and carbohy-
drate content (50.46%). The pretreated carbohydrate-rich microal-
gal biomass was also successfully used for butanol fermentation
with good performance, demonstrating the potential of using this
microalgal strain to develop an integrated process for swine
wastewater treatment and bio-butanol production.
Acknowledgements
This work was supported by the Open Project of the State Key
Laboratory of Urban Water Resource and Environment, Harbin
Institute of Technology (HCK201607). The authors also gratefully
acknowledged the support provided by Taiwans Ministry of
13
Science and Technology under grant numbers 106-3113-E-006-
011, 106-3113-E-006-004-CC2, and 104-2221-E-006-227-MY3.
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