Short Communication

Bioaugmentation of cyanide-degrading microorganisms

in a full-scale cokes wastewater treatment facility
Donghee Park
a
, Dae Sung Lee
b,
*
, Young Mo Kim
a
, Jong Moon Park
a,
*
a
Advanced Environmental Biotechnology Research Center, School of Environmental Science and Engineering/Department of Chemical Engineering,
Pohang University of Science and Technology, Pohang 790-784, South Korea
b
Department of Environmental Engineering, Kyungpook National University, Daegu 702-701, South Korea
Received 24 November 2006; received in revised form 11 March 2007; accepted 17 March 2007
Available online 21 May 2007
Abstract
To enhance biological removal eciency of total cyanides, bioaugmentation was applied to a full-scale cokes wastewaters treatment
process. After a laboratorial-scale cultivation (up to 1.2 m
3
) of a cyanide-degrading yeast (Cryptococcus humicolus) and unidentied cya-
nide-degrading microorganisms, the microbial consortium was inoculated into a uidized-bed type process (1280 m
3
), and then enriched
for two months with a huge supply of glucose, KCN and other nutrients. Target wastewater was euent of a biological pre-denitrica-
tion process for treating cokes wastewater, and contained about 14 mg/L of total cyanides in the form of ferric cyanide. This may be a
rst or rare report on the full-scale bioaugmentation of specialized-microorganisms. However, continuous operation of the full-scale cya-
nides-degrading bioprocess showed poor removal eciency than expected owing to poor settling performance of microbial ocs, slow
biodegradation rate of ferric cyanide and lack of organic carbon sources within the wastewater. Therefore, there is a need for further
studies on how to solve these operating problems in full-scale bioaugmentation approach.
2007 Elsevier Ltd. All rights reserved.
Keywords: Bioaugmentation; Cokes wastewater; Cyanide; Cyanides-degrading microorganism; Cyanides-degrading bioprocess
1. Introduction
Since cokes wastewater generated from coals coking
process of steel industries contains various toxic com-
pounds such as ammonia, thiocyanate, phenols and cya-
nides in high concentration range, it has been considered
as the most toxic one to be treated before being discharged
into environments (Li et al., 2003; Vazquez et al., 2006).
Among various processes capable of treating cokes waste-
water (Lee and Park, 1998; Ning et al., 2005; Yun et al.,
1998), a biological pre-denitrication process has been pre-
ferred in Korea because of its simplicity and economic ben-
ets (Kim et al., 2007).
The pre-denitrication process is a single-sludge system
with recycle of nitried euent and consists of two distinct
microbial reactions under anoxic followed by oxic condi-
tions (Fig. 1). In anoxic condition, heterotrophic denitrify-
ing bacteria convert nitrite and nitrate into nitrogen gas
using organic carbon sources, thus most of organic pollu-
tants are removed in this step. Additionally, very toxic free
cyanide can be removed to some degree by anaerobes
(Lewandowski, 1984). In oxic condition, autotrophic nitri-
fying bacteria convert ammonia into nitrite and nitrate,
meanwhile autotrophic thiocyanate-degrading bacteria
convert thiocyanate into ammonia, sulfate and bicarbonate
(Paruchuri et al., 1990). These successive microbial reac-
tions have completely removed most of toxic compounds
within the cokes wastewater (Table 1). However, euent
from the pre-denitrication process has contained consid-
erable amount of cyanide compounds (14 mg/L), which
must be legally removed below 1 mg/L in Korea (Note that
0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.03.027
*
Corresponding authors. Tel.: +82 53 950 7284; fax: +82 53 950 6579
(D.S. Lee); tel.: +82 54 279 2275; fax: +82 54 279 2699 (J.M. Park).
E-mail addresses: daesung@knu.ac.kr (D.S. Lee), jmpark@postech.
ac.kr (J.M. Park).
Available online at www.sciencedirect.com
Bioresource Technology 99 (2008) 20922096
a post chemical treatment process has satisfactorily
removed the residual cyanide compounds below the regula-
tion level, but numerous costs has been paid to purchase
chemical agents).
Bioaugmentation of specialized-microorganisms has
been known to be a powerful tool to enhance the removal
eciency of recalcitrant and/or toxic compounds (Van
Limbergen et al., 1998). The specialized-microorganisms
include indigenous or allochthonous wild-types or geneti-
cally modied organisms. In spite of several successes of
bioaugmentation in laboratorial-scale below 10 L (Head
and Oleszkiewicz, 2004; Jianlong et al., 2002; Saravanane
et al., 2001), its application to a full-scale wastewater treat-
ment process has rarely been tried due to the risk of irre-
coverable process failure by unexpected operating
problems. For this reason, there are few or no reports on
full-scale bioaugmentation of specialized-microorganisms
(>1000 m
3
).
In this study, a cyanide-degrading yeast, Cryptococcus
humicolus MCN2 (Kwon et al., 2002), and unidentied cya-
nide-degrading microorganisms were augmented in a full-
scale cokes wastewater treatment facility to enhance
removal eciency of cyanide compounds.
2. Experimental
2.1. Cyanides-degrading microorganisms and culture medium
A heterotrophic cyanide-degrading yeast, C. humicolus
MCN2, and unidentied cyanide-degrading heterotrophs
were used for full-scale bioaugmentation in this study. C.
humicolus MCN2 (KCTC 8708P) was obtained from a cul-
ture collection (KCTC, Korea) and has been known to uti-
lize high concentration (up to 65 mM) of KCN and
K
2
Ni(CN)
4
as a nitrogen source (Kwon et al., 2002).
Unidentied cyanide-degrading heterotrophs were isolated
from activated sludge of a full-scale cokes wastewater treat-
ment facility in a steel company, Korea (Fig. 1). A mineral
medium for isolation and enrichment was composed of 3 g
glucose, 0.5 g K
2
HPO
4
2H
2
O, 0.1 MgSO
4
7H
2
O, 0.02 g
FeSO
4
7H
2
O and 0.01 g CaCl
2
2H
2
O per liter of deion-
ized-water. Nitrogen source was supplied in the form of
KCN at a concentration of 100 mg-CN/L. All chemicals
were 9899% purity and purchased from Samchun
(Korea).
2.2. A full-scale cyanides-degrading bioprocess
A full-scale cokes wastewater treatment process is com-
posed of a biological pre-denitrication process (7930 m
3
),
a novel uidized bed type process (called as the Bio-SAC
process) (1280 m
3
) and chemical process (510 m
3
), and
has treated about 65 m
3
of cokes wastewater per hour
(Fig. 1). The Bio-SAC process was a udized-bed type
pre-denitrication process using 712 mm sized, activated
carbon coated, and waste tire-driven carrier. The content
of carrier within eight aeration tanks of the Bio-SAC pro-
cess was about 20% (v/v). The main function of the Bio-
SAC process and chemical process was to remove nitrogen
compounds remained in euent of the pre-denitrication
process. Because the pre-denitrication process had
showed satisfactory removal eciency of total nitrogen
(Table 1), the Bio-SAC plant was chosen as a space for
full-scale augmentation of cyanides-degrading microorgan-
isms in this study. Before augmentation of cyanides-
degrading microorganisms, activated sludge present in the
Bio-SAC plant was completely removed, i.e., residual
sludge content within it was nearly zero. Because of di-
culty in removing or exchanging the carriers, however, it
was remained within the Bio-SAC plant during our exper-
iment. In the beginning of our study, the existence of car-
rier was expected to enhance microbial activity owing to
the formation of biolm on its surface.
2.3. Procedure for full-scale bioaugmentation
Bioaugmentation was conducted in four steps as fol-
lows: (i) C. humicolus and cyanide-degrading microorgan-
isms were co-cultivated in a 5 L jar fermenter (BioTron,
Korea) with a 3 L of working volume at 25 C; (ii) The
microbial consortium was dividedly transferred into
100 L of 12 tanks (polyethylene) and high-densely culti-
vated for 7 weeks through batch culture where new med-
ium was supplied every week; (iii) 1.2 m
3
of highly dense
culture was then transferred into four aeration tanks
(320 m
3
of total working volume) of the Bio-SAC plant.
Augmentation was conducted under batch system for
Table 1
Characteristics of euent from a full-scale pre-denitrication process
COD Phenols Total
N
NH

4
NO

2
NO

3
SCN

Total
CN
300 ND
a
60 ND
a
3 40 ND
a
14
Total
organic
carbon
Inorganic
carbon
PO
3
4
SO
2
4
F

Cl

Fe
3+
pH
80 25 3 1000 90 1200 5
b
7.2
a
ND means Not Detected.
b
Other metals existed below 0.1 mg/L.
Fig. 1. Schematic diagram of a full-scale cokes wastewater treatment
process.
D. Park et al. / Bioresource Technology 99 (2008) 20922096 2093
29 d with supply of nutrients (1 kg/m
3
glucose, 50 g/m
3
KCN and other nutrients) per 46 days; and nally (iv)
full-scale cyanides-degrading bioprocess (i.e., 1280 m
3
)
was operated at 60 m
3
/h of internal recycle without inow-
ing of target wastewater. In addition, periodical supplies of
nutrients (0.5 kg/m
3
glucose, 25 g/m
3
KCN and other
nutrients) were conducted for 27 d. Table 2 shows adding
times and adding amounts of nutrients during full-scale
bioaugmentation period. During augmentation of cya-
nides-degrading microorganisms, temperature was 25
35 C (from April to October). Mixed liquor suspended
solid (MLSS) was used as an indicator of concentration
of cyanides-degrading microorganisms in this study.
2.4. Operation of the full-scale cyanides-degrading
bioprocess
After enough enrichment of cyanides-degrading micro-
organisms, the Bio-SAC plant was continuously operated
with inow (10 m
3
/h) of target wastewater, i.e., the eu-
ent of pre-denitrication process. Sludge return rate and
internal recycle rate were 30 m
3
/h and 60 m
3
/h, respec-
tively. Air was enough supplied in eight aeration tanks,
i.e., dissolved oxygen content was above 2 mg/L. During
the bioprocess operation, the euents were sampled to
analyze total cyanides concentration.
2.5. Analytical methods
Colorimetric method using a spectrophotometer
(GENESYS TM 5, Spectronic Inc.) was used to measure
total cyanides concentration after distillation (Meeussen
et al., 1992a). The detection limit of this method was
0.01 mg/L.
3. Results and discussion
3.1. Full-scale augmentation of cyanides-degrading
microorganisms
Cyanides-degrading microorganisms were enriched
through two steps cultivation in laboratorial-scale, and
then augmented in the full-scale Bio-SAC plant. Full-scale
augmentation of cyanides-degrading microorganisms was
also done through two steps cultivation: these microorgan-
isms were rstly cultivated in four aeration tanks (320 m
3
)
of the Bio-SAC plant under batch culture system for 29 d,
and then in the whole process (1280 m
3
) with internal
recycle system for 27 d. During the full-scale augmentation,
cyanides-degrading microorganisms could use KCN as a
nitrogen source for growth and remove it to below
0.1 mg/L. Fig. 2 shows concentration of cyanides-degrad-
ing microorganisms during two steps augmentation in
full-scale. When 1.2 m
3
of laboratory-cultivated culture
(5000 mg/L) was added into four aeration tanks, concen-
tration of sludge in these tanks was below 25 mg/L. How-
ever, sludge concentration increased by periodical supply
of glucose, KCN and other nutrients, and reached to
1203 mg/L in 29 d. To spread cyanides-degrading microor-
ganisms over the whole Bio-SAC plant, the euent of this
plant was recycled at a liquid ow rate of 60 m
3
/h, resulting
in the decrease of sludge concentration in the aeration
tanks to 200 mg/L. Additional periodical supplies of
nutrients for 27 d, however, recovered the concentration
Table 2
Adding time and adding amounts of nutrients during full-scale augmentation
Day 1 2 3 4 5 6 7 8 9 10 Total
3rd augmentation 4th augmentation
0 6 14 16 22 28 32 36 42 49
Glucose (kg) 320 320 120 360 360 360 480 640 640 560 4160
KCN (kg) 16 18 3 21 21 18 30 30 30 30 217
K
2
HPO
4
2H
2
O (kg) 32 16 0 6 3 3 4 8 8 8 88
MgSO
4
7H
2
O (kg) 10 10 2 6 3 3 4 8 8 8 62
FeSO
4
7H
2
O (kg) 4 4 3 3 3 0 2 4 0 0 23
CaCl
2
2H
2
O (kg) 2 2 2 1 1 1 2 4 4 4 21
Time (day)
0 5 10 15 20 25 30 35 40 45 50 55 60
M
L
S
S

(
m
g
/
L
)
0
200
400
600
800
1000
1200
1400
3rd augmentation
4th augmentation
1 2 3 4 5 6
7 8 9 10
Fig. 2. Concentration of cyanides-degrading microorganisms in aeration
tanks during full-scale augmentation. Arrows represent adding times of
nutrients (Table 2).
2094 D. Park et al. / Bioresource Technology 99 (2008) 20922096
of cyanides-degrading microorganisms to 1231 mg/L
(Fig. 2).
3.2. Continuous operation of the full-scale
cyanides-degrading bioprocess
After the full-scale augmentation of cyanides-degrading
microorganisms for about two months, the removal perfor-
mance of the full-scale cyanides-degrading bioprocess was
examined with the euent of pre-denitrication process.
The euent contained about 14 mg/L of cyanide com-
pounds, but no toxic compounds such as free cyanide, phe-
nol, thiocyanate and ammonia owing to the complete
biodegradation of these compounds under the anoxic fol-
lowed by the oxic conditions (Table 1). The full-scale bio-
process was continuously operated with inow (10 m
3
/
h) of the wastewater, and sludge return rate and internal
recycle rate were 30 m
3
/h and 60 m
3
/h, respectively. As
the cyanides-containing wastewater was owed, euent
concentration of total cyanides increased and reached to
6.2 mg/L in 6 d (Fig. 3). Moreover, the concentration of
activated sludge within aeration tanks decreased to below
400 mg/L because of serious washing out of it from the
sludge settling tank. This unexpected result was due to
poor settling performance of the sludge. Obviously, 3
6 mm sized microbial ocs were formed during the full-
scale augmentation, but showed poor settling performance
in continuous operation of the bioprocess. In addition, tire-
driven carrier was not proper for cyanides-degrading
microorganisms. To make matters worse, violent bubbling
of air disturbed the formation of biolm on carriers sur-
face. Finally, inux of the wastewater was intercepted at
6 d, but its removal eciency was still worse than expected.
These results implied that there might be other causes of
the poor removal eciency of total cyanides by the aug-
mented sludge.
3.3. Cause of poor removal eciency of the full-scale
cyanides-degrading bioprocess
Microbial degradation of free cyanide (KCN), nickel
cyanide (K
2
Ni(CN)
4
) and ferric cyanide (K
3
Fe(CN)
6
) by
the augmented sludge was examined by batch experiments
with a mineral medium containing 3 g/L of glucose. 20 mg/
L of free and nickelcyanides were completely degraded
by the sludge, especially 100 mg/L of free cyanide in 48 h.
However, ferric cyanide was less biodegradable than others
(data not shown). Thermodynamically, free cyanide forms
very stable complexes with iron, cobalt and gold (Ghosh
et al., 1999; Kjeldsen, 1999; Meeussen et al., 1992b). Cokes
wastewater contained cyanides in the forms of free cyanide
and ferric cyanide, since it contained only iron in the form
of ferric ion, but other metals below 0.1 mg/L (Table 1).
Fe
3
6CN

FeCN
3
6
Log K 43:9 1
It is well known that free cyanide is very toxic to microor-
ganisms, but ferric cyanide is essentially nontoxic except
UV-irradiating condition (Kjeldsen, 1999). In spite of its
acute toxicity, it has been reported that various aerobes
and anaerobes can easily and rapidly degrade free cyanide
(Akcil, 2003). However, ferric cyanide is resistant to bio-
degradation due to its thermodynamic stability under
UV-absent condition. For these reasons, only ferric cya-
nide was remained in the euent of the pre-denitrication
process (Table 1). That is to say, poor cyanides removal
eciency of the full-scale bioprocess might be due to slow
degradation rate of ferric cyanide by cyanides-degrading
microorganisms.
To examine the eect of carbon source on biodegrada-
tion of cyanides, batch experiment with the augmented
sludge and the target wastewater was also conducted with
or without glucose. Without external carbon source, the
removal eciency of total cyanides by the sludge was only
38%. However, 3 g/L of glucose enhanced it to 75% (data
not shown). Since the wastewater was euent of pre-deni-
trication process, it contained low concentration of
organic carbon sources (Table 1). Therefore, there is a need
for the supply of external organic carbon source into the
full-scale cyanides-degrading bioprocess.
3.4. Feasibility test for use of phenol as external carbon
source
Cokes wastewater contains about 200 mg/L of phenol,
thus it may be used as external carbon source for the
full-scale cyanides-degrading bioprocess. Fig. 4 shows total
cyanides concentration in the aeration tanks according to
supply amount of cokes wastewater. 1 m
3
/h of cokes waste-
water enhanced removal eciency of total cyanides. How-
ever, 2 m
3
/h of cokes wastewater increased total cyanides
concentration because of the additional supply of total cya-
nides (>25 mg/L) within it. Finally, cokes wastewater itself
Time (Day)
0 2 4 6 8 10 12 14 16 18
T
o
t
a
l

c
y
a
n
i
d
e
s

c
o
n
c
e
n
t
r
a
t
i
o
n

(
m
g
/
L
)
0
2
4
6
8
10
12
14
Influent
Effluent
Stop inflowing
Fig. 3. Concentration of total cyanides in the euent of a full-scale
cyanides-degrading bioprocess.
D. Park et al. / Bioresource Technology 99 (2008) 20922096 2095
may be used as organic carbon source for cyanides-degrad-
ing microorganisms.
4. Conclusions
To enhance biological removal eciency of total cya-
nides, cyanides-degrading microorganisms were augmented
in a full-scale cokes wastewater treatment facility. Through
long time augmentation and a huge use of nutrients, cya-
nides-degrading microorganisms were enriched in full-scale
and acclimated to cyanides-containing wastewater, i.e.,
euent of pre-denitrication process. It may be a rst or
rare report on full-scale bioaugmentation of specialized-
microorganisms. However, continuous operation of the
full-scale cyanides-degrading bioprocess showed poor
removal eciency than expected owing to poor settling
performance of microbial ocs, slow biodegradation rate
of ferric cyanide, and lack of organic carbon sources within
the wastewater.
Without doubt, bioaugmentation of specialized-micro-
organisms is a powerful tool to enhance removal e-
ciencies of recalcitrant and/or toxic compounds, but its
full-scale application seems to be not easy due to unex-
pected operating problems, as indicated in this study.
Therefore, there is a need for further studies on pilot- or
full-scale bioaugmentation to solve these operating
problems.
Acknowledgements
This work was nancially supported by the Korea Sci-
ence and Engineering Foundation through the Advanced
Environmental Biotechnology Research Center (AEBRC)
at Pohang University of Science and Technology. This
work was also supported by the ET edu-innovation Project
of Ministry of Environment in 2006.
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Time (day)
0 10 20 30 40
T
o
t
a
l

c
y
a
n
i
d
e
s

c
o
n
c
e
n
t
r
a
t
i
o
n

(
m
g
/
L
)
1
2
3
4
S
u
p
p
l
y

a
m
o
u
n
t

o
f

c
o
k
e
s

w
a
s
t
e
w
a
t
e
r

(
m
3
/
h
)
0
1
2
3
Fig. 4. Concentration of total cyanides in the euent of a full-scale
cyanides-degrading bioprocess with supply of cokes wastewater.
2096 D. Park et al. / Bioresource Technology 99 (2008) 20922096