Bioresource Technology 211 (2016) 15

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

Comparative study of wastewater treatment and nutrient recycle

via activated sludge, microalgae and combination systems
Liang Wang a, Jinli Liu a, Quanyu Zhao a, Wei Wei a, Yuhan Sun a,b,
a
CAS Key Laboratory of Low-Carbon Conversion Science and Engineering, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China
b
ShanghaiTech University, 319 Yueyang Road, Shanghai 200031, China

h i g h l i g h t s

Selected microalgae and activated sludge were co-cultured for wastewater treatment.

Chlorella sp. cultured with activated sludge in light showed the best performance.

Chlorella sp. contained biomass had higher C/H/N content compared with sludge.

PCR-DGGE analysis found two types of rhizobacteria be beneficial in consortium.

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

Article history: Algal-bacterial synergistic cultivation could be an optional wastewater treatment technology in temper-
Received 15 February 2016 ate areas. In this study, a locally screened vigorous Chlorella strain was characterized and then it was used
Received in revised form 5 March 2016 in a comparative study of wastewater treatment and nutrient recycle assessment via activated sludge
Accepted 8 March 2016
(AS), microalgae and their combination systems. Chlorella sp. cultured with AS in light showed the best
Available online 12 March 2016
performance, in which case the removal efficiencies of COD, NH3-N and TP were 87.3%, 99.2% and
83.9%, respectively, within a short period of 1 day. Algal-bacterial combination in light had the best set-
Keywords:
tleability. Chlorella sp. contained biomass, could be processed to feed, fertilizer or fuel due to the
Nutrient recycle
Activated sludge
improved quality (higher C/H/N) compared with sludge. PCR-DGGE analysis shows that two types of rhi-
Microalgae zobacteria, namely, Pseudomonas putida and Flavobacterium hauense were enriched in sludge when cul-
Rhizobacteria tured with algae in light, serving as the basics for artificial consortium construction for improved
Wastewater treatment wastewater treatment.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
Entering into a low-carbon era, energy-saving with low CO2
generating and nutrient recycling wastewater treatment processes
are in huge demand. Current prevailing treatment process, namely,
activated sludge (AS) process, has some drawbacks from the
above-mentioned perspective. For instance, ammonium needs to
undergo full nitrification with subsequent denitrification for com-
plete removal of nitrogen, which requires a lot of aeration and
extra carbon source. Meanwhile, nitrogen is excreted into the
atmosphere with some NOx generated at the same time, which
contributes to a significant portion of the greenhouse effect (Wu
Corresponding author at: CAS Key Laboratory of Low-Carbon Conversion
Science and Engineering, Shanghai Advanced Research Institute, Chinese Academy
of Sciences, Shanghai 201210, China.
E-mail address: sunyh@sari.ac.cn (Y. Sun).
et al., 2009; Beuckels et al., 2015; Yan et al., 2016). The AS system
needs to undergo frequent switch between anoxic and aerobic con-
ditions for biological nutrient removal (Hatamoto et al., 2016). In
conclusion, AS process requires extra energy and chemicals to ful-
fill the task of nutrient removal and is not quite stable at nitrogen
and phosphorus removal. Moreover, the value-added recycle mea-
sures of nitrogen and phosphorus is hardly realized through AS,
while AS is often disposed by drying and final landfill (Silva
et al., 2013).
Metabolism of nitrogen and phosphorus by microorganisms
alters their existence forms, eliminating them from the liquid
phase, the process of which is very different in microalgae from
bacteria consortia in AS. Basically, approximately all nitrogen is
taken into the algal biomass for protein synthesis and phosphorus
is partially absorbed while partially precipitated if pH is above cer-
tain level (Min et al., 2011; Kim et al., 2015; Beuckels et al., 2015).
In this way, the exogenous organic carbon source usually required

http://dx.doi.org/10.1016/j.biortech.2016.03.048
0960-8524/ 2016 Elsevier Ltd. All rights reserved.
2 L. Wang et al. / Bioresource Technology 211 (2016) 15
for denitrification in the AS process is dispensable, whereas peri-
odic supplementary CO2 is usually inevitable in microalgae
cultivation.
Algal-bacterial synergistic wastewater treatment technology
could be traced back to 1950s special stabilization pond designed
by Oswald, also named high rate algae pond (HRAP) (Oswald et al.,
1967). The technology optimized the pond depth and mixing to
enhance the growth of native algal strains for significant improve-
ment of organics, nitrogen and phosphorus removal. The HRAP sys-
tem is designed to be simple and economic in infrastructure and
operation, and the functional algal species had not been manually
selected prudently, which limited the propagation of this technol-
ogy in wastewater treatment.
In this study, a locally screened vigorous Chlorella strain was
firstly characterized and then it was used in the comparative study
of wastewater treatment and nutrient recycle assessment via acti-
vated sludge, microalgae and their combination. The removal effi-
ciencies of major nutrients, including COD, N and P were
compared, the impact of symbiotic cultivation of Chlorella with
AS on the final biomass physiological characteristics and microbial
community structure evolution is also evaluated.
2. Materials and methods
2.1. Algal strain isolation, characterization and inoculum preparation
The strain of Chlorella sp. was isolated in a local ditch with lon-
gitude and latitude of N: 31430 4000 and E: 121300 800 . Axenic cul-
ture was rendered by serial transfers on solid BG-11 plate. The
seed was cultured at room temperature (26 2 C) under cool
white fluorescent lamps with 120 lmol photons m2 s1 continu-
ous lighting condition, in air-lift glass columns using BG-11 med-
ium. The inoculum were harvested during the exponential
growth phase by centrifugation at 5000 rpm for 10 min. Then the
cells were washed three times with deionized water and used for
inoculation in comparative study. The activated sludge was
obtained from the primary settling tank of a local municipal
wastewater treatment plant.
2.2. Comparison experiment of wastewater treatment by algae,
activated sludge and their combination
Chlorella sp. and activated sludge were used to treat an artificial
wastewater. The artificial wastewater contained (per liter): NaAc
1400 mg; NH4Cl, 200 mg; FeSO47H2O, 0.55 mg; CaCl2, 6 mg;
KH2PO4, 50 mg; MgSO47H2O, 66 mg; H3BO3, 2.86 mg; MnCl24H2O,
1.86 mg; ZnSO47H2O, 0.22 mg; Na2MoO42H2O, 0.39 mg;
CuSO45H2O, 0.08 mg; Co(NO3)26H2O, 0.05 mg. Both light and dark
conditions were applied in the comparison experiment. The 5
experimental treatments for wastewater remediation were
designed: (1) Chlorella sp. alone were cultured in light, (2) Chlorella
sp. mixed with activated sludge were cultured in light, (3) Chlorella
sp. alone were cultured in dark, (4) Chlorella sp. mixed with
activated sludge were cultured in dark, (5) activated sludge alone
were cultured in dark. Treatments with only algae or only sludge
had an initial algae or bacteria biomass of 1.0 g/L, while the mixed
treatments contained 0.5 g/L of algae plus 0.5 g/L of bacteria. The
experiments were lasted for 46 h, till when all the major nutrients
had been exhausted. Mixed liquid samples for all treatments were
taken at 0 h, 6 h, 22 h, 30 h and 46 h. Chlorella sp. and activated
sludge were cultured in a 1000 ml Erelenmeyer flask with a
working volume of 500 ml and bubbled with the compressed air
by an air stone at the bottom of the flask. The light intensity
applied was 200 lmol photons m2 s1 and all groups were
cultured at 26 2 C in triplicates.
2.3. Analysis methods
The biomass growth was determined by the gravimetric
method after drying. In detail, the cellulose acetate membranes
(pore size 0.45 lm, d = 5 cm) were dried overnight at 105 C and
weighted after they were cooled down to the room temperature.
Cultures with a certain volume (V) were filtered onto a pre-
weighted membrane (W0) and washed twice with distilled water
to remove the residual salts. Then the membrane with biomass
was weighted (W1) after drying at 105 C for 24 h. The dry weight
of the biomass was calculated as: DW = (W1  W0)/V, where V is
the sampling volume (mL) and W0 and W1 the initial and final
membrane weights (mg). CODs, NH4-Ns and TPs of the water sam-
ples were measured by HACH DR2800 standard methods. The con-
tents of total carbon, hydrogen and nitrogen in the dried biomass
was analyzed by a CHNS Analyzer (Thermo scientific Flash 2000).
Sludge volume index (SVI) and media pH were analyzed as per
standard methods (APHA, 2005).
2.4. PCR-DGGE analysis of sludge contained samples
Polymerase chain reaction (PCR) was performed on the DNA
extracts of the original AS samples, and 3 of 46-h AS contained
samples using universal primers with subsequent analysis by
denaturing gradient gel electrophoresis (DGGE), cloning, sequenc-
ing and phylogenetic analysis in order to access the sludge micro-
bial community evolution in the treating process. In details, total
bacterial DNA was extracted from 250 mg each sample centrifuged
at 8000 rpm for 5 min. DNA was extracted by using Mobio Power-
soil Isolation Kit (MO BIO, Inc., CA, US) according to the manufac-
turers protocol. PCR was performed with the primers 357F-GC
and 518R, located at the V3 region of the 16S rDNA genes. PCR con-
ditions used were 2 min of denaturation at 94 C, followed by 30
cycles of 30 s at 94 C, 30 s at 56.4 C, and 30 s at 72 C. A final
extension step at 72 C for 5 min was performed to finish the reac-
tion. DGGE was performed with 8% (wt/vol) acrylamide gels con-
taining a linear chemical gradient ranging from 40% to 60%
denaturant with 100% defined as 7 M urea and 40% formamide.
Gels were run for 16.5 h at 80 V with the Dcode Universal Mutation
System (Bio-Rad Laboratories, Hercules, CA, US). Major bands were
excised for identification of bacterial species. Sequence analyses
were done by using the BLAST database (National Center for
Biotechnology Information, www.ncbi.nlm.nih.gov). Sequence
alignments were performed by using the clone Manager 7.1
software.
3. Results and discussion
3.1. Characterization of the isolated Chlorella strain
In order to initiate the comparison experiment, it was necessary
to locate a suitable microalgal strain which has all the merit prop-
erties, such as: (a) fast-growing, (b) fast reduction of major nutri-
ents, (c) tolerant to high temperature, pH or irradiation, and (d)
able to establish harmony with wastewater indigenous bacteria
and beneficial to nutrient recycle. In the preliminary tests this
competent strain of Chlorella was screened out, which has 99% sim-
ilarity to Chlorella sorokiniana (NIES collection #2173) according to
18s rDNA sequencing and BLAST homology comparison. The strain
was demonstrated to be superior to other isolated or purchased
strains in both growth and nutrient removal capabilities on various
culture media and wastewaters and could contain nitrogen as high
as 10% of its dry weight. Plenty of literature have confirmed that
Chlorella is often the predominant species in algal wastewater
treatment pond systems (Garca et al., 2000; de Godos et al.,
 L. Wang et al. / Bioresource Technology 211 (2016) 15 3

2009; Hernndez et al., 2013) and highly effective in removal of 60 Chlorella sp.
organics, nitrogen and phosphorus (Wang et al., 2010a,b). AS
Chlorella sp.+light
Chlorella sp.+AS
Chlorella sp.+AS+light
3.2. Comparison on the removal of COD, N and P in wastewater under 40

NH3-N (mg/L)
different conditions

As shown in Fig. 1, COD concentrations of each group decreased

rapidly in the starting period, especially in the first 6 h. The AS con-
20
tained treatments showed to have better COD removal efficiency
than Chlorella sp. alone treatments, while Chlorella sp. cultured in
dark was the least efficient one for COD reduction within a certain
limited period of time. AS is mainly composed of bacterial clusters,
belonging to the species of Actinobacteria, b-Proteobacteria and Fir- 0
0 10 20 30 40 50
micutes (Hashimoto et al., 2014), which can utilize acetate as the
carbon source to proliferate rapidly as long as the oxygen supply Time (h)
in the system is sufficient. Chlorella sp. alone cultured in dark could Fig. 2. NH3-N consumption in the five groups. j Chlorella sp. alone were cultured
utilize organic carbon only in its heterotrophic mode, and com- in dark. d Activated sludge alone were cultured in dark. N Chlorella sp. alone
pared with AS, microalgae could not reproduce themselves as were cultured in light. . Chlorella sp. mixed with activated sludge were cultured
quickly as prokaryotic bacterial cells, thus they could not assimi- in dark. Chlorella sp. mixed with activated sludge were cultured in light.

late carbon source at the speed as AS did. Chlorella sp. alone cul-
tured in light could grow faster than those cultured in dark
through the mixotrophic mode, obtaining extra energy for carbon
assimilation from photons (Yan et al., 2012), thus COD could be
reduced in a more efficient way when light was supplied. Basically,
COD was reduced faster in groups containing AS than groups con-
taining Chlorella sp. The residual CODs showed little difference
between groups after 22 h because the organics were almost con-
sumed completely either by AS or microalgae.
As shown in Fig. 2, NH3-N content decreased significantly in
each group except AS and flattened out after 20 h. Nitrification is
a slow process and the results indicate that this AS did not contain
a significant portion of effective nitrification bacteria. In light or in
dark, Chlorella sp. alone works well to remove the NH3-N, while in
light the NH3-N removal rate was much faster than that in dark,
which corresponds to the COD removal rates under the same con-
ditions, indicating the utilization of carbon and nitrogen for
microalgae was performed synergistically and light could serve
as the accelerator for nutrient uptake process related to the mixo-
trophic mode of microalgae (Garci et al., 2005). Finally, Chlorella sp.
alone cultured in dark, Chlorella sp. alone cultured in light, and
Chlorella sp. mixed with activated sludge cultured in light could
completely remove NH3-N.
Activated sludge alone almost has no effect on the removal of
phosphorus as shown in the Fig. 3, indicating that the
phosphorus-accumulating bacteria were not enhanced in the AS
and phosphorus removal by AS need periodic cycles of anaerobic
and aerobic conditions, so it is not beneficial to apply AS in a single
batch system for phosphorus removal. Chlorella sp. grown in light
was the most efficient way for phosphorus removal, which could
remove around 12 mg/L P completely within 6 h. Chlorella sp. cul-
tured with AS in light could fast reduce P to below 1.0 mg/L in 6 h
but slowly release small amount of P afterwards, which should be
attributed to the partially unstable fixation of P by AS. Chlorella sp.
grown in dark fixed phosphorus slower than those cultured in light
or with AS, performing in the way the same as for COD and NH3-N.
Considering removal of all the three major nutrients in wastewa-
ter, i.e., COD, NH3-N and TP, Chlorella sp. cultured with AS in light
showed the best performance, in which case the removal efficien-
cies of COD, NH3-N and TP were 87.3%, 99.2% and 83.9%, respec-
tively, within the period of 1 day.

1500 Chlorella sp.

Chlorella sp. 15 AS
AS Chlorella sp.+light
Chlorella sp.+light Chlorella sp.+AS
1200 Chlorella sp.+AS Chlorella sp.+AS+light
Chlorella sp.+AS+light 12

900
COD (mg/L)

9
TP (mg/L)

600
6

300
3

0
0 10 20 30 40 50 0
Time (h) 0 10 20 30 40 50
Time (h)
Fig. 1. COD consumption along with time in the five groups. j Chlorella sp. alone
were cultured in dark. d Activated sludge alone were cultured in dark. N Fig. 3. TP consumption in the five groups. j Chlorella sp. alone were cultured in
Chlorella sp. alone were cultured in light. . Chlorella sp. mixed with activated dark. d Activated sludge alone were cultured in dark. N Chlorella sp. alone
sludge were cultured in dark. Chlorella sp. mixed with activated sludge were were cultured in light. . Chlorella sp. mixed with activated sludge were cultured
cultured in light. in dark. Chlorella sp. mixed with activated sludge were cultured in light.
4 L. Wang et al. / Bioresource Technology 211 (2016) 15

3.3. Physiological characteristics of the mixed biomass under different
conditions
Study on physiological characteristics of the mixed biomass
includes, settleability, as evaluated by SVI, and major elemental
composition. An SVI that does not exceed 150 mL/g is an indicator
of good settling properties of the sludge in wastewater treatment
systems (Janczukowicz et al., 2001). The SVI measurement was
performed for sludge contained groups in order to examine the set-
tleability of sludge or sludge-algae mixtures. The SVIs were 76.80,
57.14, and 42.55 mL/g, for sludge alone, Chlorella sp. cultured with
AS in dark and Chlorella sp. cultured with AS in light, respectively,
indicating that the algae addition in the treatment system could
improve the comprehensive settleability. Especially when cultured
in light the SVI could be lowered down 44.6% compared with the
activated sludge alone system. Gravity sedimentation is hardly
applied for unicellular microalgae, while the physiochemical tech-
niques, such as flocculation, filtration, or centrifugation have to be
applied in order to harvest microalgae (Christenson and Sims,
2011). Current research demonstrated that when combined with
activated sludge in the wastewater treatment system, microalgae
could settle down through natural bio-flocculation with sludge
within 30 min, which would greatly reduce the algae harvesting
cost since it accounts for around 30% of algae cultivation cost if
the harvesting of microalgal biomass is realized by dissolved air
flotation, subsequent centrifuge and final membrane ultrafiltration
in large-scale cultivation (Richardson et al., 2014). However, mix-
ing with sludge may reduce the high-value uses of the algal bio-
mass, e.g., pharmaceutical or nutraceutical uses.
The elemental (C/H/N/S) analysis was done for the harvested
mixed biomass in different treatments and the results are shown
in Table 1. Generally, Chlorella sp. cultured in dark or light, mixed
with AS or not, had higher nitrogen content, which is due to the
different cellular composition of microalgae and sludge. Algae
could function as a nutrient sink while sludge function as a conver-
tor for major nutrients. For instance, sludge respires organic carbon
to CO2, and converts dissolved nitrogen to dinitrogen gas through
nitrification and denitrification. AS had the lowest nitrogen, as well
as carbon and hydrogen, because AS is characterized by more ash
content (Choi et al., 2015). Therefore, AS usually has very limited
further processing and utilization value, and could only be fer-
mented to methane or landfilled (Lin et al., 2014) in practice. How-
ever, Chlorella sp. contained biomass, could be processed to feed,
fertilizer or fuel due to the improved quality (higher C/H/N) com-
pared with sludge, which brings additional value to wastewater
treatment and should serve as a more environmentally-friend car-
rier for nutrient (N & P) recycle.
3.4. The microbial community evolution analysis
The DGGE profiles of bacteria community sampled from the
original AS sample and the 46-h AS contained samples are summa-
rized in Table 2.
In order to gain more insight into microbial structure evolution
when cultured with algae or not, thirteen predominant species
extracted from the DGGE bands were sequenced and matched
based on the 16S rDNA gene library results (Table 2). Band 1, 2,
7, 8 and 9, are identified as Fluviicola taffensis, Pseudomonas
panipatensis, Bacillus persicus, Sphingopyxis terrae, and Citrobacter
freundii, respectively, were retained in prolonged sludge treatment
but diminishing when cultured with algae, under light or not. Band
3 and 5 are identified as Pseudomonas putida and Flavobacterium
hauense, which stayed unchanged in sludge and sludge with algae
in dark, but were enriched in sludge cultured with algae in light,
indicating that there were some mutual benefits between these
microorganisms and Chlorella, when cultured together in light,
which could serve as an effective consortium for nutrient removal.
P. putida and Flavobacterium are classified as rhizobacteria, which

Table 1
Elemental (C/H/N/S) analysis of harvested mixed biomass in different treatments.

N (%) C (%) H (%) S (%)

Chlorella sp. 10.01 0.06 48.00 0.42 6.83 0.01 0.13 0.01
AS 4.93 0.23 28.35 0.62 4.23 0.21 0.19 0.07
Chlorella sp. + AS 8.24 1.19 38.24 2.94 5.47 0.32 0.12 0.01
Chlorella sp. + light 5.51 0.06 46.90 1.03 6.71 0.20 0.09 0.01
Chlorella sp. + AS + light 6.76 0.60 40.22 3.13 5.70 0.45 0.10 0.03

Table 2
DGGE 16S rDNA band identifications.

Band Run Genbank accession No. Closest relatives (%Sequence similarity d) Classe
1a 2 3 4
1

b


c
NR_074547.1 98 Fluviicola taffensis strain DSM 16823
2





NR_044209.1 100 Pseudomonas panipatensis strain Esp-1
3




NR_074739.1 100 Pseudomonas putida F1 strain F1
4



NR_074264.1 100 Caulobacter sp. K31 strain K31
5




NR_109662.1 98 Flavobacterium hauense strain BX12
6




NR_1256 91.1 100 Pseudocitrobacter anthropi strain C138
7





NR_109140.1 99 Bacillus persicus strain B48
8





NR_113727.1 100 Sphingopyxis terrae strain NBRC 15098
9





NR_117752.1 100 Citrobacter freundii strain LMG 3246
10




NR_074240.1 100 Polymorphum gilvum strain SL003B -26A1
11




NR_117752.1 100 Citrobacter freundii strain LMG 3246
12






NR_074240.1 99 Polymorphum gilvum strain SL003B-26A1
13





NR_114293.1 98 Flavihumibacter petaseus strain NBRC 106054
a
Corresponding to A, B, C, D, E in the DGGE profile.
b
Existence with a higher intensity under the condition.
c
Existence with a lower intensity under the condition.
d
Percent values represent similarities between the associated DGGE band sequence and the closest match sequence from GenBank.
e
The phylotypes were assigned to phyla based on the BLAST database.
 L. Wang et al. / Bioresource Technology 211 (2016) 15
can colonize a wide range of plants and provide plant growth pro-
moting effects through nitrogen fixation, phosphate solubilization,
and production of phyto-hormones, such as gibberellin and indol-
3-acetic acid. (Kang et al., 2014; Egamberdieva et al., 2015;
Pishchik et al., 2002). From the DGGE profiling it can be inferred
that the harmony between these rhizobacteria and higher plants
also exists between them and microalgae. Band13 is identified as
Flavihumibacter petaseus, which was depressed when cultured with
algae in dark whereas enhanced when cultured with algae in light,
as shown in sludge treatment alone, indicating that this kind of
bacteria might be used in synergistic cultivation with algae in mix-
otrophic mode. The specific mechanism to explain the relationship
between the nutrient removal efficiency and the sludge-Chlorella
mutual interaction under different settings is yet to be uncovered.
Shannon factor, which is one of the many factors used to
describe the microbial diversity, is calculated for AS contained
samples in different conditions. After 46 hs wastewater treatment
process, the Shannon factor of the AS stays unchanged of around
2.85 for AS alone and AS with Chlorella sp. in light treatments,
whereas decreases when AS was cultured with algae in dark with
a value around 2.69, indicating that microalgae might pose some
negative effect on some phyla of bacteria in the heterotrophic
mode and become more as nutritional competitors to bacteria
(Pearson et al., 1987). Green microalgae have very different meta-
bolic pathways in mixotrophic and heterotrophic modes (Liang
et al., 2009), while mixotrophic mode might stimulate the synthe-
sis of the necessary cytochromes for the respiratory chain (Neilson
and Lewin, 1974), which could explain why there were different
microbial community evolutions in sludge cultured in dark com-
pared with that cultured in light. The above results indicated that
the diversity of bacterial communities in different conditions could
be affected by introducing microalgae, which could be an explana-
tion of different treatment performance in these settings. Further
identification and isolation of the potential beneficial synergistic
bacterial strains and respective co-culture with microalgae may
be helpful for artificial consortium construction and better opti-
mization of the treatment system.
4. Conclusions
In this study, a locally screened vigorous Chlorella strain was
characterized and used in a comparative study of wastewater
treatment via activated sludge, microalgae and their combination
systems. Chlorella sp. cultured with AS in light showed the best
performance, in which case the removal efficiencies of COD,
NH3-N and TP were 87.3%, 99.2% and 83.9%, respectively, within
a short period of 1 day. Chlorella sp. contained biomass had higher
C/H/N content compared with sludge. PCR-DGGE analysis shows
that two types of rhizobacteria, namely, P. putida and F. hauense
were enriched in sludge when cultured with algae in light.
Acknowledgements
This study is supported by National Natural Science Foundation
of China (No. 51208305), Science and Technology Commission of
Shanghai Municipality Program (No. 14DZ1203800) and Shanghai
Pujiang Program (No. 13PJ1407600).
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