Bioresource Technology 225 (2017) 58–66

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

Feasibility of using a microalgal-bacterial consortium for treatment of

toxic coke wastewater with concomitant production of microbial lipids
Byung-Gon Ryu a,⇑, Jungmin Kim b,c, Jong-In Han d, Ji-Won Yang e,f
a
Freshwater Bioresources Utilization Bureau, Nakdonggang National Institute of Biological Resources, 137, Donam 2-gil, Sangju-si, Gyeongsangbuk-do 37242, Republic of Korea
b
Future Environmental Research Center, Korea Institute of Toxicology, Jinju 52834, Republic of Korea
c
Human and Environmental Toxicology Program, Korea University of Science and Technology, Daejoen 34113, Republic of Korea
d
Department of Civil and Environmental Engineering, KAIST, 291 Daehakno, Yuseong-gu, Daejeon 305-701, Republic of Korea
e
Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehakno, Yuseong-gu, Daejeon 305-701, Republic of Korea
f
Advanced Biomass R&D Center, KAIST, 291 Daehakno, Yuseong-gu, Daejeon 305-701, Republic of Korea

h i g h l i g h t s

The feasibility of microalgal-based treatment of coke wastewater was evaluated.

Activated sludge was mainly responsible for the degradation of phenol and COD.

Greater fatty acid accumulation occurred with greater dilutions of coke wastewater.

RDA-NMDS clarified the effects of wastewater dilution and activated sludge.

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

Article history: This study examined the feasibility of using an algal-bacterial process for removal of phenol and NH+4-N
Received 1 September 2016 from differently diluted coke wastewater with simultaneous production of biomass. Under illumination,
Received in revised form 28 October 2016 microalgal-bacterial (MSB) cultures performed complete phenol degradation at all dilutions of coke
Accepted 5 November 2016
wastewater while sole microalgal culture (MSA) degraded a maximum of 27.3% of phenol (initial concen-
Available online 9 November 2016
tration: 24.0 mg L1) from 5-fold diluted wastewater. Furthermore, the MSB culture had the highest rate
of NH+4-N removal (8.3 mg L1 d1) and fatty acid production (20 mg L1 d1) which were 2.3- and 1.5-
Keywords:
fold higher than those observed in the MSA cultures, probably due to decreases in toxic organic pollu-
Coke wastewater
Microalga
tants. Multivariate analyses indicated that co-cultivation of activated sludge was directly correlated with
Activated sludge the elevated removals of phenol and NH+4-N. In the presence of sludge, adequate dilution of the coke
Nitrogen removal wastewater can maximize the effect of bacteria on NH+4-N removal and biomass production.
Statistical analysis Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Coke effluent generated from coal coking, coal gas purification,
and recovery processes in steel manufacturing is a significant toxic
industrial waste product which contains high levels of toxicants
such as phenolic compounds, polynuclear aromatic hydrocarbons
(PAHs), cyanide compounds, and ammonium nitrogen (NH+4-N)
(Kim et al., 2009). An activated sludge process (ASP), consisting
of aerobic nitrification and anaerobic denitrification, is a reliable
method that is commonly used to treat this coke wastewater
(Kim et al., 2011, 2008). However, coke effluent generally contains
various toxic chemicals, so a sudden failure of the ASP can occur
⇑ Corresponding author.
E-mail address: tesia@nnibr.re.kr (B.-G. Ryu).
(Kim et al., 2009). For instance, 1–2 mg L1 of free cyanide (or even
less) in the form of HCN or CN can significantly inhibit microbial
activities involved in the oxidation of nitrogen by ammonium-
oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)
(Kim et al., 2009, 2008). It is typical that nitrifying bacteria are
rather hard to maintain in a stable way; and therefore ASP-based
nitrogen removal is often unsatisfactory (Kim et al., 2011).
Microalga-based wastewater treatment can be a promising
alternative. It is, first of all, because microalgae have the ability
to efficiently remove nitrogenous pollutants such as NH+4, NO 2,
NO 3 , and urea by simple uptake under photoautotrophic or hetero-
trophic conditions. In addition, many algal species can accumulate
fatty acids that could be converted into biodiesel at much higher
than the oleaginous land crops (Matamoros et al., 2015). Besides,
there exist species that can tolerate potentially toxic compounds

http://dx.doi.org/10.1016/j.biortech.2016.11.029
0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
 B.-G. Ryu et al. / Bioresource Technology 225 (2017) 58–66
such as PAHs, biocides, surfactants, cyanide, and phenolic com-
pounds, and some even degrade these chemicals via metabolic-
dependent or -independent pathways (de-Bashan and Bashan,
2010; Gurbuz et al., 2004; Klekner and Kosaric, 1992; Luo et al.,
2014; Muñoz et al., 2003; Ryu et al., 2014a). A hydraulic retention
time (HRT) of more than 2 days (longer than needed for a bacteria-
based treatment process) must be maintained to prevent the
‘‘wash-out” of algal biomass during wastewater treatment. How-
ever, a recently proposed algal-based process is economically fea-
sible because it consumes almost 30-fold less electricity
(0.02 kWh m3) than that a typical ASP (0.6 kWh m3)
(Matamoros et al., 2015).
Algae and bacteria grown symbiotically could enhance the
applicability of microalga-based methods for treatment of wastew-
ater that contains both organic and inorganic pollutants (Posadas
et al., 2013; Su et al., 2012; Zhao et al., 2014). In this symbiosis,
the O2 released by algal photosynthesis is utilized by aerobic-
heterotrophic bacteria to mineralize organic compounds, and bac-
terial respiration provides CO2 as a carbon source to the algae.
Thus, the advantages are cost-efficient aeration, efficient removal
of pollutants, and mitigation of greenhouse gas emissions (Su
et al., 2012). Moreover, algae can concomitantly remove nutrients
and pathogens (including viruses) (Munoz et al., 2003). Algal-
bacterial flocs, which often occur during wastewater treatment,
can promote subsequent downstream processes for biomass har-
vesting by simple gravity sedimentation without use of flocculat-
ing agents (Munoz and Guieysse, 2006; Ryu et al., 2014b). Owing
to these advantages, previous studies have examined cooperative
biosystems of algal-bacterial consortia using freshwater or marine
microorganisms to treat various wastewaters such as raw munici-
pal effluent, sludge concentrates, saline wastewater, and landfill
leachate, by use of different culture strategies (e.g. raceway ponds,
aerobic or anoxic-aerobic photobioreactors, and fixed-bed photo-
bioreactors) (Babatsouli et al., 2015; de Godos et al., 2014;
Posadas et al., 2013; Su et al., 2012; Zhao et al., 2014).
The results of these previous reports led to the hypothesis that
microalga alone or algal-bacterial cultures can catabolically
degrade toxic organic pollutants, mainly phenol, and simultane-
ously assimilate the NH+4-N from coke wastewater, and that aerobic
sludge from a sewage wastewater treatment plant can be used as
an effective bacterial seed (Guo and Chen, 2015; Ryu et al.,
2014b). However, there is limited information on the microalga-
mediated processes for treatment of coke wastewater.
The aim of the present study was to assess the feasibility of a
microalga-mediated process to simultaneously treat phenol and
NH+4-N from the coke effluent and simultaneously produce microbial
lipids for potential use as biofuels. More specifically, this study exam-
ined the effects of the wastewater dilution ratio and the presence of
AS on the removal of phenol, soluble chemical oxygen demand
(SCOD), and NH+4-N by the green alga, Scenedesmus quadricauda, and
the cellular growth and fatty acid production of this alga under illumi-
nation. Multivariate statistical analysis, redundancy analysis (RDA),
and a map of nonmetric multidimensional scaling (NMDS) were also
employed to interpret the relationships of wastewater dilution and
AS inoculum with removal of phenol, removal of NH+4-N, algal growth,
and production of cellular fatty acids.
2. Materials and methods
2.1. Preparation of microbial seed culture and coke wastewater
2.1.1. AS from a sewage plant and the green alga Scenedesmus
quadricauda
AS was collected from an aerobic reactor installed in a local
sewage treatment plant in Daejeon, Republic of Korea. The aerobic
59
AS was centrifuged at 6000g for 10 min, and then the bio-pellet
was used as an aerobic bacterial seed. The green microalga
S. quadricauda (AG10003) was obtained from the culture collection
of the Korean Research Institute of Bioscience and Biotechnology
(KRIBB, Daejeon, Republic Korea), and cultivated in a Tris-acetate
phosphate (TAP) medium consisting of: Beijerinck’s solution
(7.5 mM NH4Cl, 0.35 mM CaCl22H2O, and 0.4 mM MgSO47H2O),
phosphate buffer solution (0.69 mM Na2HPO4 and 0.45 mM KH2-
PO4), Hunter’s trace metal solution (134 lM Na2EDTA2H2O,
77 lM ZnSO47H2O, 184 lM H3BO3, 26 lM MnCl24H2O, 18 lM
FeSO47H2O, 7 lM CoCl26H2O, 5 lM CuSO45H2O, and 8 lM
(NH4)6Mo7O244H2O), and Tris-acetate solution (20 mM Tris and
17 mM glacial acetic acid). These cultures were exposed to contin-
uous fluorescent light (120 lmol m2 s1) on a rotary shaker
(120 rpm) at 25 °C.
2.1.2. Preparation of coke wastewater with different dilutions
Coke wastewater was collected from a full-scale wastewater
treatment facility in the coke plant of a steel manufacturing com-
pany in the Republic of Korea. The sample was filtered through
0.22-lm membrane filter (Sartorius Stedim Biotech, Göttingen,
Germany) and stored in sterilized bottles at 4 °C. Table 1 shows
the physicochemical parameters of the coke wastewater. The
major pollutants are phenol, soluble COD, and NH+4-N, typical of
coke wastewater. For experiments, the coke wastewater was
diluted with deionized water (DIW) to obtain solutions with
100%, 80%, 60%, 40%, and 20% coke wastewater.
2.1.3. Inoculation of microbial seeds into the diluted coke wastewater
The seed cultures of S. quadricauda (grown in TAP medium) and
the aerobic AS were centrifuged at 6000g for 10 min, and the
resulting pellets were inoculated into the prepared wastewater.
The initial concentration of each biomass was adjusted to
200 mg L1 of volatile suspended solids (VSS). Two different cul-
tures – S. quadricauda alone (MSA) and algal-bacterial (MSB) mixed
cultures – were cultivated in 250-mL baffled flasks (150-mL work-
ing volume) with diluted coke wastewater at 25 °C on a rotary sha-
ker (120 rpm), under continuous illumination (120 lmol m2 s1
of fluorescent light) with 2% CO2 (v/v) at a rate of 0.25 gas volume
per liquid volume per minute (vvm). Coke wastewater has a very
low concentration of phosphate (<0.1 mg L1), an essential element
for algal photosynthesis. Thus, phosphate salts (0.69 mM Na2HPO4
and 0.45 mM KH2PO4) were added into the MSA and MSB cultures
with different dilutions of coke wastewater. All batch tests were
performed in triplicate.
Table 1
Physicochemical properties of the coke wastewater used in this study.
Characteristics Values
pH 7.8
Total solid (TS) (mg L1) 737.0 ± 7.8
Total suspended solid (TSS) (mg L1) 260.0 ± 14.1
Volatile solid (VS) (mg L1) 340.0 ± 28.3
Volatile suspended solid (VSS) (mg L1) 10.0 ± 0.0
SCODcr (mg L1) 2860 ± 42.4
Phenol (mg L1) 429 ± 9.2
Ammonium (NH+4)-N (mg L1) 114.4 ± 4.5
Nitrate (NO 3 )-N (mg L
1
) 6.9 ± 0.2
Nitrite (NO2 )-N (mg L
1
) ND
Free cyanide (CN) (mg L1) 6.1 ± 0.8
Fluoride (F) (mg L1) 104.3 ± 0.9
1
Sulfate (SO2
4 ) (mg L ) 999.6 ± 4.4
Sodium (Na+) (mg L1) 1998.2 ± 52.9
All data are mean values from triplicate experiments, and standard deviations are
given rounded off to two decimal places.
ND: not detected.
60 B.-G. Ryu et al. / Bioresource Technology 225 (2017) 58–66
2.2. Analytical methods
2.2.1. Volatile suspended solids (VSS)
The concentrations of VSS before and after coke wastewater
treatment were measured. A 20-mL sample was taken and then
passed through a glass microfiber filter (GF/C; Whatman, Kent,
UK), which was prepared by pre-ignition at 550 °C for 30 min to
volatilize residual organic matter on the filter. The filtered sample
was dewatered at 105 °C for 2 h and then ignited at 550 °C for
30 min. The VSS was considered to be the difference in weight
between the dried and ignited sample.
2.2.2. Soluble organic carbon and nitrogen
The supernatant of wastewater samples (prepared by centrifu-
gation at 6000g for 5 min) was filtered with 0.22-lm membrane
filters (Sartorius Stedim Biotech, Göttingen, Germany), and the sol-
uble components in the filtrates were measured. Ion chromatogra-
phy, (883 Basic IC plus; Metrohm AG, Herisau, Switzerland)
equipped with a cationic column (Metrosep C4 – 150/1.0;
Metrohm AG) and an anionic column (Metrosep A Supp 5 –
150/1.0; Metrohm AG), was used to determine the concentration
of cations (NH+4 and Na+) and anions (F, NO  2
2 , NO3 , PO4 , and
2 
SO4 ). Concentrations of SCOD, phenol, and cyanide (CN ) were
determined by water analysis kits (Humas; HS-COD-MR, HS-
Phenol, HS-CN, Republic of Korea). The removal efficiency of each
pollutant (phenol, SCOD, and NH+4-N) was measured as:
Removal efficiency ð%Þ ¼ ðCi  Ct Þ=Ci  100
where Ci and Ct are the concentrations of a pollutant at the initial
stage and after the indicated time, respectively.
2.2.3. Chlorophyll-a
The concentration of chlorophyll-a was measured in the bio-
pellets of 2-mL culture samples which were centrifuged at 6000g
for 10 min. All hydrophobic pigments were extracted from pre-
pared bio-pellets by addition of dimethyl sulfoxide (DMSO) at
65 °C for 60 min. After extraction, the supernatant (containing
chlorophyll-a in DMSO) was separated by centrifugation and a
UV–vis spectrophotometer was used to measure absorbance at
649 nm (A649) and 665 nm (A665). The concentration of
chlorophyll-a was estimated as (Ryu et al., 2014b):
Chlorophyll-a ¼ 12:47  A665  3:62  A649
2.2.4. Fatty acid methyl esters (FAMEs)
The composition of fatty acids were determined by the analyz-
ing fatty acid methyl esters (FAMEs) after the transesterification of
extracted lipids. Each cell biomass was prepared by centrifugation
at 6000 g for 10 min and then freeze-dried at 52 °C for 3 days.
Cellular lipids from 10 mg of the lyophilized biomass were
extracted by a mixture of chloroform and methanol (2:1, v/v),
according to the modified Folch’s method (Ryu et al., 2014a). A
subsequent transesterification of extracted lipids was performed
by a mixture of methanol (the reactant) and sulfuric acid (the cat-
alyst) at 100 °C for 20 min in a heating block. Afterwards, DIW was
added to the reaction mixture and then further centrifuged at
6000 g for 5 min. After the centrifugation, the organic phase (bot-
tom layer) were separated from water phase (upper layer) and
then the FAMEs in the organic phase were determined by a gas
chromatograph (HP5890; Agilent, CA, USA) equipped with a flame
ionization detector (FID) and an INNOWAX capillary column (Agi-
lent; 30 m/0.32 m/0.5 lm). The temperature of the gas chro-
matograph (GC) column was gradually raised at 15 °C min1
from 50 to 200 °C, where it was retained for 2 min. Finally, the
temperature of GC column was steadily increased at 10 °C min1
from 200 to 250 °C.
2.2.5. Statistical analysis
To compare the statistical differences between MSA and MSB
culture in terms of removals of phenol, SCOD, NH+4-N, changes in
chlorophyll-a content, biomass production based on DCW, pro-
duced FAMEs and compositions of fatty acids, a two-tailed t-test
was employed. Pearson correlation coefficient and one-way analy-
sis of variance (ANOVA) were also analyzed to evaluate quantita-
tive association with statistical significance among
environmental factors (wastewater dilution and presence of aero-
bic bacteria), removals of phenol and NH+4-N, and FAMEs
production.
Redundancy analysis (RDA) is an iterative analysis in which
reciprocal averaging/correspondence is based on a linear and direct
gradient-constrained ordination method (Leps and Smilauer,
2003). RDA was employed using CONACO software (version 4.5;
Microcomputer, Ithaca, NY, USA) to determine the statistical rela-
tionship between environmental factors (AS dilution and presence
of aerobic bacteria) and their measured parameters as dependent
factors for NH+4 removal, growth of microalga, chlorophyll-a con-
tent, and production of fatty acids. This analysis examined the sig-
nificance of constrained ordination methods based on a Monte
Carlo permutation test to provide a simple graphical summary
(Leps and Smilauer, 2003).
NMDS is a multivariate statistical ordination method that can
identify non-parametric monotonic correlation based on dissimi-
larities in the item-item matrix, and the Euclidean distances
between items. The location of each item in the low-dimensional
space was also determined (Fromin et al., 2002). NMDS concen-
trates on the response data of one lane to one point in a two-
dimensional plane, thereby clustering the samples by an
unweighted pair group method with arithmetic mean (UPGMA)
clustering analysis based on the Jaccard similarity coefficient
(Fromin et al., 2002). PC-ORD software (version 5; MjM Software,
Wagga Wagga, NSW, Australia) was used to examine the correla-
tions of environmental factors with measured parameters, and to
characterize each sample by simple clustering.
3. Results and discussion
3.1. Degradation of phenol and soluble COD
Fig. 1 shows the time-dependent changes in phenol and SCOD
in MSA and MSB cultures grown with different concentrations of
coke wastewater and the statistical significances of the differences
were summarized in Supplementary Table 1. These results show
negligible phenol degradation by the MSA culture for 60–100%
coke wastewater. The MSA culture removed 27.3 ± 0.3% of the phe-
nol when the coke wastewater concentration was 20%, and
6.1 ± 0.1% when concentration was 40% (Fig. 1a, b, and c). Although
the degradation efficiency of phenol was low, a higher dilution of
coke wastewater with reduced toxicity correlated with better phe-
nol degradation. Thus, the meta-cleavage pathway for microalgal
catabolism of phenol seemed to be retarded by toxicants present
in the coke wastewater (Pinto et al., 2002). In contrast to phenol
degradation, the MSA cultures did not degrade SCOD, even when
the concentration of the coke wastewater was 20%
(Fig. 1d, e, and f). It may be that some fragments derived from
the incomplete decomposition of phenol or phenol metabolites
were detected as SCOD (Luo et al., 2014).
The MSB cultures successfully degraded phenol at all coke
wastewater concentrations, and removed at least 99.0 ± 0.0% of
the phenol (Fig. 1a, b, and c). This implies that some aerobic-
heterotrophic bacteria in the AS contributed to the degradation
of phenol. As the MSB cultures degraded phenols, there were cor-
responding declines in the SCOD with overall degradation levels
 B.-G. Ryu et al. / Bioresource Technology 225 (2017) 58–66 61

Fig. 1. Effect of coke wastewater concentration on percent degradation of phenol (top) and SCOD (bottom) at 48 h (a, d), 96 h (b, e), and 144 h (c, f) by cultures of the
microalga S. quadricauda alone (MSA) and by a microalgal-bacterial mixed cultures (MSB).

of 43.8 ± 0.1–49.1 ± 0.0% depending on the coke wastewater con-
centration (Fig. 1d, e, and f); the results were statistically signifi-
cant at p < 0.05 (Supplementary Table 1).
3.2. Removal of NH+4-N
Fig. 2 shows the time-dependent changes of NH+4-N in MSA and
MSB cultures with different concentrations of coke wastewater and
the statistical significances of the differences were summarized in
Supplementary Table 1. NH+4-N is considered one of the most ener-
getically preferred nitrogen sources by microalgae, in contrast to
the oxidized forms of nitrogen (NO 
2 and NO3 ) which must be
reduced via intracellular reductases (Markou and Georgakakis,
2011). However, at 45 h the MSA and MSB cultures did not take
up any nitrogen when the coke wastewater concentration was
60% or more, probably due to toxic effects. When the concentration
of the coke wastewater was 40% or less, the MSA and MSB cultures
successfully removed all or nearly all of the nitrogen by 144 h.
Thus, when coke wastewater is sufficiently diluted, MSA and
MSB cultures can completely remove NH+4-N, irrespective of the
presence of AS.
The initial concentration of NH+4-N depends on the concentra-
tion of the coke wastewater. Table 2 summarizes the total amount
of consumed NH+4-N and their uptake rates by MSA and MSB cul-
tures. These results show that the MSB cultures had a maximum
uptake rate that was 2.3-fold greater than that of the MSA cultures;
the results were statistically significant at p < 0.05 (Supplementary
Table 1). Thus, co-inoculation with AS appears to promote more
rapid NH+4-N removal. The poorer uptake of NH+4-N by the MSA cul-
ture is presumably due to oxidative stress triggered by various
toxic chemicals such as phenols, nitrogenous heterocyclic com-
pounds, PAHs, anilines, napthalenols, and oxygenic heterocyclic
compounds that can form reactive oxygen species (ROS) from coke
wastewater (Li et al., 2003). These ROS can inhibit photosynthetic
electron transfer and the formation of NADPH, leading to decreased
photosynthesis (Qian et al., 2009). Alternatively, there may have
been disruption of enzymatic reactions by fluoride (F), a toxic
anion in coke wastewater, which can target Mg2+-adenosine

Fig. 2. Effect of coke wastewater concentration on percent degradation of NH+4 at 48 h (a), 94 h (b), and 144 h (c) in MSA and MSB cultures.
62 B.-G. Ryu et al. / Bioresource Technology 225 (2017) 58–66

triphosphatase (Mg2+-ATPase) and Ca2+-ATPase of microalgae, also

Ammonium (NH+4)-nitrogen uptake rates (mg L1 d1) = DCa/DT, where DCa and DT indicated the consumed amount of NH+4-nitrogen (mg L1) at a defined time and the interval of cumulative cultivation time in days,
decreasing photosynthetic electron transfer (Camargo, 2003; Rai
et al., 1998). A previous study reported that the levels of CN
and thiocyanate (SCN) present in ASP are likely to have negligible
Total uptake rate
(mg L1 d1)c
effects on microalgae (Gurbuz et al., 2004; Ryu et al., 2014a,b).
During nitrogen removal, no NO 
2 or NO3 (which could be gener-

6.1 ± 0.0d
4.7 ± 0.1
5.1 ± 0.0
4.6 ± 0.0

8.3 ± 0.0
+
ated from the oxidation of NH4) were detected (data not shown),
probably because toxic compounds in the coke wastewater inhib-
ited most of the ammonium-oxidizing bacteria or nitrifying bacte-
Total consumed NH+4-

ria in the aerobic AS (Kim et al., 2011, 2009; Ryu et al., 2014b,
2015b).
On the other hand, the elevated rate of NH+4 removal in the MSB

Total uptake rate (mg L1 d1) = (Ci  Ce)/(T  T0), where Ci and Ce are the initial and final concentration of NH+4-N, and the difference between T and T0 is the cultivation time in days.
cultures could also be explained by detoxification due to bacterial
24.2 ± 0.0d
27.5 ± 0.1
28.3 ± 0.5
49.7 ± 0.0
30.7 ± 0.0
N (mg)b

mineralization of organic pollutants or dilution of toxic compounds

(Munoz and Guieysse, 2006; Su et al., 2012). Apart from the
reduced toxicity due to the actions of the bacteria and/or dilution
effects, there could also be a symbiotic relationship of the microal-
13.3 ± 0.0e

6.7 ± 0.2
5.3 ± 0.0

1.0 ± 0.0

gae and bacteria (often classified as plant growth-promoting bacte-

144

ria, PGPB) in the AS (or pure strain) (Gonzalez and Bashan, 2000;
ND

Ryu et al., 2015b). However, the molecular mechanism of this rela-

20.6 ± 1.4e
10.6 ± 0.2e

tionship remains unknown.

9.2 ± 0.8e
1.5 ± 1.1

5.4 ± 0.4

The values indicated the maximum levels of the time-dependent NH+4-N uptake rate during their growth under different contents of coke wastewater.

Considering the nitrogen uptake rate by microalgae previously

reported for other types of wastewater (Table 3), the application
94
Changes in the rate of ammonium (NH4+)-nitrogen uptake over time by MSA and MSB cultures grown in different concentrations of coke wastewater.

of an algal-bacterial process appears to have high potential for

6.7 ± 0.0e

removal of NH+4-N from toxic coke effluent. A subsequent hetero-

3.0 ± 0.3

trophic mode, supplied by an additional cheap carbon source such

ND
ND
ND
45

as short chain fatty acids generated from an anaerobic digestion

process or volatile fatty acids (VFAs), can significantly enhance
Time
MSB

the removal of NH+4-N and also be used for biomass production

(h)

(Ryu et al., 2015a). This enhanced process with a combined hetero-

trophic mode would make this process an even more attractive
Total consumed NH+4-N (mg) = Ci  Ce, where Ci and Ce are the initial and final concentration of NH+4-N, respectively.

option.
Total uptake rate
(mg L1 d1)c

3.3. Microbial growth and cellular fatty acids

6.1 ± 0.0d
2.2 ± 0.1
2.8 ± 0.0

6.6 ± 0.0
3.0 ± 0.0

3.3.1. Changes in cellular chlorophyll-a

Total consumed NH+4-N (mg) and total uptake rate (mg L1 d1) were calculated at 94 h of operation.

Chlorophyll-a content was measured as an indicator of microal-

gal growth and cell viability (Fig. 3) and the statistical significances
Total consumed NH+4-

of the differences were summarized in Supplementary Table 1.

These results show the MSA cultures had a lower (even decreased)
chlorophyll-a content compared to the initial state when the coke
wastewater concentration is 60% and above. However, the MSB
24.2 ± 0.0d
12.8 ± 0.7
16.8 ± 0.2

39.8 ± 0.1
18.1 ± 0.0
Ammonium (NH+4)-nitrogen uptake rates (mg L1 d1)a

N (mg)b

cultures had 2-fold more chlorophyll-a at 40% wastewater concen-

tration and 1.63-fold greater chlorophyll-a at 20% wastewater con-
centration; the results were statistically significant at p < 0.05
(Supplementary Table 1). The greatest chlorophyll-a concentration
7.3 ± 0.3e

8.9 ± 0.0e
2.6 ± 0.1
0.5 ± 0.0

was achieved in 40% coke wastewater in MSB cultures. These

144

ND

results are consistent with microalgal uptake of NH+4-N for use in

chlorophyll synthesis. Another plausible explanation could be
10.7 ± 0.3e
8.1 ± 0.8e

6.2 ± 0.0e

related to the presence of significant ROS at high coke wastewater

8.3 ± 0.3

concentrations; previous research indicated that ROS can attack

ND
94

chlorophyll-a and inhibit photosynthesis (Cardozo et al., 2002).

In this stressful condition, changes in cellular morphology can also
2.4 ± 0.1
6.1 ± 0.3

occur (Supplementary Fig. 1) (Pinto et al., 2002). Therefore, consid-

ND: The NH+4-nitrogen was not decreased.
ND
ND
ND
45

erable efforts, such as reducing the toxicant loading rate or pre-

treatment, must be implemented to maintain or increase
Time

microalgal photosynthesis and cell viability and assure stable

MSA

(h)

nitrogen removal, especially when there are co-existing toxicants.

3.3.2. Changes in biomass production and cellular fatty acids

100% (no dilution)
Contents of coke

Fig. 4a shows the effect of coke wastewater concentration on

Type of culture

the production of volatile suspended solids (VSS) in MSA and

wastewater

respectively.

MSB cultures after 144 h and the statistical significances of the dif-
ferences were summarized in Supplementary Table 2. These results
Table 2

80%
60%
40%
20%

indicate no significant changes in the VSS when the wastewater

a

e
c
b

concentration was 60% or more in MSA cultures, possibly due to

 B.-G. Ryu et al. / Bioresource Technology 225 (2017) 58–66 63

Table 3
Comparison of nitrogen removal rates by different algal species in different wastewaters.

Type of wastewater Species Light-dark Operation Nitrogen source Initial Removal Removal rate References
cycle time (d) concentration efficiency (%) (mg L1 d1)
(mg L 1)
Municipal Coelastrum 12–12 h 12 Total dissolved 39.7 88.0 2.9
wastewatera microporum nitrogen
Municipal Desmodesmus Continuous 7 NH+4-N 55.9 100.0 7.9
wastewater communis
(PCE)b
Municipal Desmodesmus Continuous 11 NH+4-N 84.6 100.0 7.7
wastewater communis
(PCE)b
Municipal Galdieria sulphuraria 12–12 h 7 NH+4-N 40.0 88.3 4.9
wastewater
(PCE)b
Municipal Algal-bacterial 12–12 h 8 NH+4-N 18.9 100.0 2.4
wastewater mixed cultured
(SCE)c
Dairy manurea Chlorella sp. Continuous 21 NH+4-N 178.0 100.0 8.5
Piggery wastewater Chlorella pyrenoidosa Continuous 10 NH+4-N 138.8 91.2 12.7
Coke wastewater Scenedesmus Continuous 7 (7)⁄ NH+4-N 49.7 80.2 6.6 (8.9)⁄ This study
quadricauda
Coke wastewater S. quadricauda- Continuous 7 (3.9)⁄ NH+4-N 49.7 100 8.3 (20.6)⁄ This study
bacterial culture

Values in parentheses indicate maximal NH+4-N removal rate at the specified time.
a
The wastewater was produced from an anaerobic digester.
b
PCE, primary clarifier effluent.
c
SCE, secondary clarifier effluent.
d
The mixed algal-bacterial inoculum was obtained from the second clarifier wall of the municipal wastewater plant.

Fig. 3. Change in chlorophyll-a concentration over time in different concentrations of coke wastewater in MSA cultures (a) and MSB cultures (b).

Fig. 4. Effect of coke wastewater concentration on volatile suspended solids (mg L1, a), total FAMEs content (mg g1, b), and FAMEs concentration (mg L1, c) in MSA and
MSB cultures after 144 h.
64 B.-G. Ryu et al. / Bioresource Technology 225 (2017) 58–66

inhibition of microalgal growth. In contrast, the VSS increased to at
least 1.10 g L1 in MSB cultures for all concentrations of wastewa-
ter. However, these trends were inconsistent with the observed
changes in chlorophyll-a concentration (an indicator of microalgal
growth) when the wastewater concentration was 60% or more.
Considering the discordant changes in VSS and chlorophyll-a con-
tent in MSA cultures, it seems likely that aerobic bacteria in the AS
accounted for a large portion of the total VSS in MSB cultures
because microalgal chlorophyll-a remained low under these condi-
tions. Thus, the increased level of VSS is likely due to the growth of
aerobic-heterotrophic bacteria in the AS.
Fig. 4b and c show the effect of coke wastewater concentration
on total FAMEs content and concentrations in MSA and MSB cul-
tures with after 144 h. These results show that MSA cultures gen-
erally produced slightly more fatty acids (mg g1) than MSB
cultures, although the amounts were similar to those observed in
20% coke wastewater. Based on previous findings, this may be
because bacteria present in the AS, which produce a relatively
lower long-chain fatty acids compared to microalga, account for
a large portion of the total biomass in MSB cultures. However,
when the FAMEs content is normalized to biomass concentration,
the accumulated FAMEs content was greater in MSB cultures; at
a wastewater concentration of 20%, the MSA cultures produced
93.6 mg L1 of FAMEs and the MSB cultures produced 130.8 mg L1
of FAMEs; the results were statistically significant at p < 0.05 (Sup-
plementary Table 2).
Table 4 shows that coke wastewater concentrations affected
total FAMEs production and the FAME profile, and the statistical
significances of the differences were also summarized in Supple-
mentary Table 3. In particular, these results indicate that total fatty
acids and relatively long-chain saturated and mono- or poly-
unsaturated fatty acids, such as steric acid (C18:0), oleic acid
(C18:1), linoleic acid (C18:2), and arachidic acid (C20:0), were neg-
atively associated higher levels of residual toxic pollutants. Basi-
cally, the synthesis of long chain saturated or unsaturated fatty
acids closely correlated with the activity of the electron transport
system and the production of ROS (Paradies et al., 2002). Unsatu-
rated fatty acids are especially susceptible to oxidative attack
because carbon-carbon double bonds can undergo peroxidation
through a chain of oxidative reactions (Cardozo et al., 2002;
Paradies et al., 2002). When levels of these long-chain unsaturated
fatty acids decrease, the lipid quality (potential use as a transport
fuel) also declines based on oxidative stability and cold-flow prop-
erties (Ryu et al., 2014b). Therefore, lowering the concentration of
ROS-producing toxicants in the coke wastewater by appropriate
dilution or pretreatment can improve the quality of the microbial
lipids that are produced.
3.4. Statistical analyses of the relationships of wastewater dilution and
co-inoculation with AS with removal of phenol and NH+4-N and
production of FAMEs
Pearson’s correlation coefficient analysis with one-way ANOVA
showed that the dilution ratio of wastewater and presence of aer-
obic bacteria were differently correlated with various independent
parameters (Supplementary Table 4): strong positive correlations
(Pearson correlation coefficient q > 0.8, dilution-NH+4-N removal:
q = 0.8646; dilution-FMAEs production: q = 0.9253; aerobic
bacteria-phenol removal: q = 0.8758), moderate positive correla-
tions (0.8 > q > 0.5, aerobic bacteria-NH+4-N removal: q = 0.5858;
aerobic bacteria-FAMEs production: q = 0.5858), and weak positive
correlation (q < 0.5, dilution-phenol removal: q = 0.1158).
RDA was employed to investigate the relationships of wastew-
ater dilution and co-inoculation of AS with removal of NH+4-N and
phenol and production of FAMEs (Fig. 5a). This analysis indicates
that the sum of all canonical eigenvalues was 0.95, indicating a sta-
tistically meaningful relationship. If a relationship is positive, the
environmental parameter has a linear response to the dependent
variable. Thus, the results indicate that removal of NH+4-N and phe-
nol were positively associated with co-inoculation of AS. In a
sequential manner, aerobic-heterotrophic bacteria in the AS first
degrade the phenol, irrespective of the concentration of coke
wastewater, so that bacterial oxidation of toxic organic matter
enhances the assimilation of the NH+4-N by the microalgae. In con-
trast, the effect of wastewater dilution on these two parameters
was not significant. These finding are similar to those observed
in the analysis of Pearson correlation (Supplementary Table 4).
The correlation between wastewater concentration and FAME

Table 4
Composition and profile of fatty acid methyl esters (FAMEs) produced by MSA and MSB cultures after coke wastewater treatment under different conditions.

Parameters Compositions of cellular fatty acids

MSA MSB
100%a 80% a
60% a
40% a
20% a
100%a 80% a
60% a
40% a
20% a

Type of Saturated fatty C14:0 1.6 ± 0.0 1.3 ± 0.1 0.4 ± 0.1 1.0 ± 0.1 – – – – 1.1 ± 0.1 0.2 ± 0.0
fatty acids (SF)
acid (%)
C16:0 47.5 ± 0.0 42.9 ± 0.0 28.1 ± 0.3 27.2 ± 0.1 30.5 ± 1.3 51.3 ± 2.3 53.5 ± 0.8 39.4 ± 2.5 25.7 ± 0.6 24.7 ± 0.3
C18:0 – – 2.9 ± 0.0 7.1 ± 0.1 2.5 ± 0.2 – – 3.9 ± 0.1 8.2 ± 0.1 3.6 ± 0.1
C20:0 – – 1.4 ± 0.1 1.4 ± 0.0 5.4 ± 0.5 – – – 2.2 ± 0.1 1.5 ± 0.0
Sub 49.1 ± 0.0 44.2 ± 0.1 32.7 ± 0.3 36.6 ± 0.1 38.3 ± 0.6 51.3 ± 2.3 53.5 ± 0.8 43.3 ± 2.3 37.1 ± 0.4 30.0 ± 0.2
total
Mono-saturated C16:1 – – 0.4 ± 0.0 1.3 ± 0.1 – 7.1 ± 0.0 11.0 ± 1.6 8.0 ± 0.1 2.7 ± 0.0 0.9 ± 0.0
fatty acids (MSF)
C18:1 28.0 ± 0.1 26.9 ± 0.0 37.7 ± 0.4 15.0 ± 0.1 43.4 ± 1.2 33.6 ± 2.3 18.2 ± 0.5 19.5 ± 0.4 23.1 ± 0.0 43.2 ± 0.0
Sub 28.0 ± 0.1 26.9 ± 0.0 38.1 ± 0.4 16.3 ± 0.2 43.4 ± 1.2 40.7 ± 2.3 29.2 ± 1.1 27.5 ± 0.3 25.8 ± 0.0 44.1 ± 0.0
total
Poly-unsaturated C18:2 – 5.7 ± 0.2 13.2 ± 0.1 16.5 ± 0.1 13.5 ± 0.4 – – 16.4 ± 0.7 3.0 ± 0.2 6.1 ± 0.0
fatty acids (PUFA)
C18:3 12.1 ± 0.1 10.7 ± 0.1 10.5 ± 0.2 20.5 ± 0.0 0.5 ± 0.0 – – – 22.3 ± 0.5 13.5 ± 0.4
Sub 12.1 ± 0.1 16.4 ± 0.1 23.7 ± 0.4 37.0 ± 0.1 14.0 ± 0.4 – – 16.4 ± 0.7 25.2 ± 0.7 19.6 ± 0.4
total
Unknown 10.9 ± 0.0 12.6 ± 0.3 5.6 ± 0.3 10.2 ± 0.1 4.4 ± 0.4 8.1 ± 0.1 17.5 ± 1.9 12.9 ± 1.3 11.9 ± 0.3 6.4 ± 0.1
FAMEs contents (mg g1) 33.0 ± 0.8 38.3 ± 0.9 40.4 ± 0.9 53.1 ± 0.8 86.0 ± 0.7 25.7 ± 1.9 28.1 ± 5.1 29.9 ± 0.5 39.0 ± 1.4 94.5 ± 1.5
FAMEs concentration (mg L1) 14.7 ± 2.0 17.6 ± 0.1 15.5 ± 0.7 38.3 ± 2.1 92.9 ± 1.7 32.6 ± 2.1 33.9 ± 0.7 32.5 ± 0.8 47.5 ± 1.6 139.8 ± 3.2
a
Content (%) of coke wastewater.
 B.-G. Ryu et al. / Bioresource Technology 225 (2017) 58–66
Fig. 5. Redundancy analysis (a) and map of nonmetric multidimensional scaling (b)
showing the relationships of independent and dependent variables in MSA and MSB
cultures.
production was positive and linear, indicating that the effect of
phenol degradation on the synthesis of cellular fatty acids was
not significant, possibly due to the presence of other residual
organic matter or inorganic compounds, such as F, that can gener-
ate oxidative stress.
The distribution patterns and similarities of samples in the MSA
and MSB cultures with different coke wastewater concentrations
were also analyzed by NMDS, and then characterized as clusters
based on the relationships of dependent and independent variables
(Fig. 5b). This analysis shows the relationships between the depen-
dent and independent variables were similar to those observed in
the RDA, supporting the validity of the RDA. The NMDS analysis
65
clearly shows the clustering of samples into 4 different groups with
respect to changes in environmental factors: MSA cultures at 100,
80, 60, and 40% coke wastewater (Group A), MSB cultures at 100,
80, and 60% coke wastewater (Group B), MSA cultures at 20% coke
wastewater (Group C), and MSB cultures at 20 and 40% coke
wastewater (Group D). Groups A and B were negatively correlated
with wastewater concentration and its corresponding dependent
factors (FAMEs and chlorophyll-a) even though the wastewater
had different dilutions. This means that the effect of wastewater
dilution at these levels on pollutant removal and biomass produc-
tion by the MSA cultures were statistically meaningless. However,
Group C, which is mainly influenced by wastewater dilution, is
close to the vector direction for production of FAMEs, and thereby
segregated from Group A. Group D is influenced by environmental
parameters and was linearly consistent with all responses, includ-
ing removal of phenol and NH+4-N, and production of chlorophyll-a
(biomass) and FAMEs. Therefore, the NMDS analysis demonstrates
that an appropriate dilution of coke wastewater can maximize the
effect of AS on pollutant removal and algal metabolism. According
to these findings, the initial concentrations of 600–1190 mg L1
SCOD and 82–170 mg L1 phenol (by dilution or controlling HRT)
can achieve better performance and a more stable operation of a
microalgal-bacterial process for treatment of coke wastewater.
The microalgae/sludge ratio must be also optimized.
4. Conclusion
This study investigated the feasibility of a microalgal-bacterial
process for treating toxic pollutants in coke wastewater. The
results indicated that the AS was mainly responsible for the com-
plete degradation of phenol and that this bacterial mineralization
of toxic organic compounds in coke wastewater led to increased
algal assimilation of NH+4-N. The findings further indicated that
appropriate dilution of coke wastewater can maximize the effect
of AS bacteria on pollutant removal and lipid production. Further
optimization of the algae/AS ratio and HRT in a continuous process
should make the proposed system a more efficient and feasible
means for treating coke wastewater.
Acknowledgement
This work was supported by the Advanced Biomass R&D Center
(ABC) of Korea via a grant funded by the Ministry of Science, ICT
and Future Planning of the Republic of Korea (ABC-2010-0029728).
This work was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korean government, Ministry of
Science, ICT and Future Planning of the Republic of Korea (No.
2012M2A8A5025996).
This work was supported by the Nakdonggnag National Insti-
tute of Biological Resources grant funded by the Ministry of Envi-
ronment, Republic of Korea.
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.2016.11.
029.
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