Bioresource Technology 176 (2015) 38–46
Contents lists available at ScienceDirect
Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
Zinc chloride as a coagulant for textile dyes and treatment of generated
dye sludge under the solid state fermentation: Hybrid treatment
strategy
Avinash A. Kadam a, Harshad S. Lade b, Dae Sung Lee a, Sanjay P. Govindwar c,⇑
a
b
c
Department of Environmental Engineering, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 702-701, Republic of Korea
Department of Environmental Engineering, Konkuk University, Seoul 143-701, Republic of Korea
Department of Biochemistry, Shivaji University, Kolhapur 416004, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Novel coagulant ZnCl2 for textile
wastewater treatment.
Coagulation was studied using UV–
vis, FTIR, FE-SEM EDX and FE-TEM.
Dye sludge decolorized under SSF
solve one of major problem of
coagulation process.
Scale up to pilot scale coagulation
reactor and composting bioreactor.
Effective toxicity removal by
combining both coagulation and SSF.
a r t i c l e
i n f o
Article history:
Received 11 September 2014
Received in revised form 24 October 2014
Accepted 27 October 2014
Available online 4 November 2014
Keywords:
Hybrid technology combining
physicochemical and biological process
Solid state fermentation
Coagulation of textile dyes
Decolorization
Composting
a b s t r a c t
Dye sludge generation is major drawback of coagulation process. Efficient hybrid technology by combining coagulation and solid state fermentation (SSF) has capacity to solve generated dye sludge problem.
Coagulation of 100 mg/L Reactive Red 120 (RR120) using ZnCl2 showed 99% color removal. Mixture of
textile dyes (MTD) and textile wastewater (TW) showed 96% and 98% ADMI (American Dye Manufacturing Institute) removal after coagulation by ZnCl2. 92% and 94% ADMI removal from MTD and TW dye
sludge and 96% decolorization of RR120 sludge was observed respectively by developed microbial consortium (DCM) in 72 h under SSF. Scale up of coagulation process by coagulation reactor (CR) having
50 L capacity operated for 30 min/cycle. CR showed average 94% ADMI removal from TW in 10 successive
cycles. Scale up of SSF composting bioreactor (CB) showed complete dye removal from dye sludge
obtained from CR (500 L of TW) in 30 days.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Textile industries are most polluting industries worldwide
(Saratale et al., 2011). Approximately, 40–65 L of textile wastewater is produced per kg of cloth and released textile wastewater
⇑ Corresponding author. Tel.: +91 231 2609152.
E-mail addresses: spg_biochem@unishivaji.ac.in, spgovindwar@rediffmail.com
(S.P. Govindwar).
http://dx.doi.org/10.1016/j.biortech.2014.10.137
0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
(TW) was containing higher concentrations of dyes (Manu and
Chaudhari, 2002). These dyes released into wastewater were found
to be adversely affecting the environment (Sanghi et al., 2006;
Tayade et al., 2007; Natarajan et al., 2011). Rapid industrialization
in developing countries releases the various pollutants which lead
to the pollution of extremely important fresh water resources (Lee
et al., 2006). Dye wastewater from textile industries was contributing significantly for water pollution due to its aesthetically improper color and toxicity to aquatic flora/fauna and also for human
A.A. Kadam et al. / Bioresource Technology 176 (2015) 38–46
beings (Lade et al., 2012). Therefore, it is adequate to take attempts
to solve the problem caused by dye wastewater.
The present wastewater treatments mainly involve physicochemical and biological methods. Physicochemical methods for
dye removal consist of coagulation, ozonation, fenton process,
membrane filtration, photocatalysis, sonication, irradiation, photochemical process, electrochemical oxidation, ion exchange and
adsorption (Saratale et al., 2011) and biological methods using bacteria, fungi, algae and plants (Kadam et al., 2014). Each method has
its own drawbacks and advantages.
Coagulation is most commercially used physicochemical process worldwide for TW treatment (Meric et al., 2005). It is mainly
because of efficacy of the process and immediate treatment of TW.
Immediate treatment was extremely necessary in consideration of
large volume of TW generated daily (Kadam et al., 2014). Various
coagulants were reported for dye removal involve pre-hydrolysed
metallic salts (polyaluminium chloride, polyaluminium ferric chloride, polyferrous sulphate and polyferric chloride), hydrolysing
metallic salts (aluminium sulphate, ferric chloride, ferric sulphate,
manganese chloride and magnesium chloride) and synthetic cationic polymers (aminomethyl polyacrylamide, polyalkylene, polyamine,
polyethylenimine,
polydiallyldimethyl
ammonium
chloride (Verma et al., 2012).
Detailed review by Verma et al. (2012) on existing coagulation
techniques for TW treatment clearly suggested that; although
coagulation as an efficient and cost effective treatment accepted
in worldwide, the novel coagulant should be addressed and studied
for direct TW containing complex mixture of textile dyes. Simultaneously, generation of secondary dye sludge is one of the major
drawbacks of the technique and very less emphasize is being given
to its further treatment (Chu, 2001; Sanghi et al., 2006). Therefore,
addressing the novel coagulants having capacity to remove dyes
from wastewater along with treatment of secondary dye sludge
generated will provide an additional insight for TW treatment.
In biological processes, the use of microorganisms for dye treatment has upper hand in concern with higher dye concentration
and treatment time (Saratale et al., 2011). But most of dye decolorization study was carried out in submerged fermentation (SmF)
conditions (Saratale et al., 2011; Lade et al., 2012). By seeing engineering and economic advantages of SSF (solid state fermentation)
over SmF, SSF found to be the best suited over SmF processes for
bioremediation of dye (Masaphy et al., 1996; Kadam et al.,
2013a,b, 2014). Agricultural wastes are mainly used as substrates
in SSF processes (Waghmare et al., 2014). Availability of agricultural wastes to treat pollutants signifies the low cost technology
which can be affordable for textile industries (Nigam et al.,
2000). Similarly composting approach, scale up of SSF was found
significant in the removal of variety of pollutants (Gunderson
et al., 1997; Laine and Jørgensen 1997; Jørgensen et al., 2000;
Canet et al., 2001; Namkoong et al., 2002). Composting approach
for TW treatment was not exploited yet, despite the fact that it
showed significant capacity to remove variety of pollutants.
Combination of different techniques enables improved performance of wastewater treatment (Lee et al., 2006). Treatment of
coagulated dye sludge under SSF conditions was novel asset for
TW treatment combining most valuable physical coagulation process and economical biological process SSF. As far as our knowledge, there is no report suggesting hybrid technology combining
coagulation and biological process SSF.
Hence, this study investigates the effect of coagulant zinc chloride (ZnCl2) for removal of an individual Reactive Red 120 (RR120),
dyes from mixture of textile dyes (MTD) and textile wastewater
(TW). As far as our knowledge, this is a first report where ZnCl2
has been used as a coagulant for textile dyes treatment in batch
scale as well as in laboratory scale reactor. Generated dye sludge
from coagulation process was decolorized under SSF conditions
39
by developed microbial consortium (DMC). Pilot scale reactors
were designed for coagulation process scale up using coagulation
reactor (CR). Scale up of biological process SSF was done using
composting bioreactor (CB). Phytotoxicity studies were carried
out for detoxification analysis.
2. Methods
2.1. Materials
All chemicals were of an analytical grade and of highest purity.
ZnCl2 and microbiological medium nutrient broth were purchased
from HiMedia Laboratories Pvt. Ltd., Mumbai, India. The textile
dyes such as Reactive Red 120 (RR120), Reactive Blue 19, Rubin
GFL, Navy Blue HE2R, Remazol Red, Remazol Black B, Red HE3B,
Orange 122, Navy Blue 2R, Blue 2RNL, Direct Red 5B, Golden yellow
HE2R and Green HE4B were obtained from local textile processing
industry, Ichalkaranji, India. Mixture of textile dyes (MTD) was
prepared in distilled water by mixing 10 mg/L of Reactive Red
120, Reactive Blue 19, Rubin GFL, Navy Blue HE2R, Remazol Red,
Remazol Black B, Blue 2RNL and Direct Red 5B dyes. The effluent
was collected in airtight plastic container. This collected effluent
was filtered by ordinary filter paper to remove large suspended
particles. The pH of the filtered effluent was adjusted to 7.0 and
stored at 4 ± 1 °C until use.
2.2. Coagulation study for ZnCl2
2.2.1. Batch scale coagulation study of textile dyes using coagulant
ZnCl2
The 100 mL of various textile dyes such as Reactive Red 120,
Reactive Blue 19, Rubin GFL, Navy Blue HE2R, Remazol Red, Remazol Black B, Red HE3B, Orange 122, Navy Blue 2R, Blue 2RNL,
Direct Red 5B, Golden yellow HE2R and Green HE4B was taken at
the concentration of 100 mg/L separately in a 250 mL Erlenmeyer
flasks. A concentration of 11 mM/L of coagulant ZnCl2 was added
to the flask. After addition of coagulant, the pH of solution was
adjusted to 10 by using the 0.5 N NaOH. This mixture was then
immediately stirred at 60 rpm for 1 min. Coagulate of dyes formed
was allowed to settle at the bottom of flask for 1 min. 3 mL clear
supernatant was removed by 1 mL micropipette and used to measure the color removal at respective maximum absorption of textile dyes. Similar process was studied for MTD and TW. In order
to optimize the pH for coagulation, the pH of the RR120 solution
and TW was adjusted to 2, 4, 6, 8, 10 and 12 after the addition of
coagulant ZnCl2 (11 mM/L) and agitated for 1 min at 60 rpm. The
effect of agitation time was studied by keeping the flask for 1, 2,
3, 4 and 5 min at 60 rpm on shaker after coagulant ZnCl2
(11 mM/L) addition and then pH was adjusted to 10. Increasing
concentration of ZnCl2 was taken as 1.83, 3.66, 5.50, 7.33, 11.0
and 14.6 mM/L to study its effect on coagulation of RR120 solution
and TW by keeping pH 10 and agitated for 1 min at 60 rpm. Various
temperatures such as 10, 20, 30, 40 and 50 °C were studied for its
effect on coagulation process with pH 10, agitation time 1 min at
60 rpm and coagulant concentration 11 mM/L.
2.2.2. Investigation of coagulation mechanism of ZnCl2
In order to investigate the possible removal of dye RR120 during
coagulation, the RR120 (100 mg/L) sludge obtained after the coagulation was filtered using Whatmann filter paper No. 42. The filtered RR120 sludge was further taken into the 100 mL distilled
water. The pH of the solution was adjusted to 7 using 0.1 N HCl.
After dissolution of the precipitate, the sample from above solution
was scanned in visible spectrophotometric region using Hitachi
40
A.A. Kadam et al. / Bioresource Technology 176 (2015) 38–46
Fig. 1. Optimization of coagulation process parameters such as [1] pH, [2] agitation time, [3] coagulant (ZnCl2) concentration and [4] temperature.
U-2800 spectrophotometer. Similarly the untreated RR120 solution was scanned in visible region of light. Investigation of possible
coagulation mechanism of MgCl2 was studied similarly by Gao
et al. (2007). Coagulation was carried out in presence and absence
of RR120, and the dried sample of coagulate with RR120 and coagulate without RR120 were analyzed to elucidate the structural differences and probable coagulation mechanism using techniques
like Fourier transform infrared spectroscopy (FTIR, PerkinElmer,
Spectrum GX & AutoImage), field emission scanning electron
microscope with energy dispersive X-ray (FE-SEM EDX, Hitachi
S-4800), field emission transmission electron microscopy
(FE-TEM, FEI, Titan G2 ChemiSTEM Cs Probe) and X ray diffraction
(XRD, Rigaku, D/Max-2500).
added to the flask and incubated for 15 d to enrich micro flora from
the soil. 0.5 g of the soil sample from above mixture was added to
the flask containing 5 g of rice bran and 10 mL of coagulated dye
sludge. The moisture content of the flask adjusted to 90% and incubated at 30 °C. Microbial consortium showing decolorization at
solid state conditions was selected and evaluated for its ability to
decolorize consistently. 0.2 g of sample from these flasks transferred to nutrient medium and allowed to grow at 30 °C for 24 h.
The obtained microflora was again confirmed for dye sludge decolorization under SSF conditions and named as DCM. The DCM was
maintained on nutrient agar slants and stored at 4 °C for further
use.
2.2.3. Environmental parameters analysis
The environmental characteristics such as total organic carbon
(TOC), chemical oxygen demand (COD) and biological oxygen
demand (BOD) of MTD and TW were studied for samples obtained
before and after the coagulation process (APHA, 1998). COD of the
textile effluent was measured using automated COD analyzer
(Spectralab CT 15, India). The total organic carbon (TOC) was measured using Hach K9 DR 2700 spectrophotometer (Hach Co., USA)
(Lade et al., 2012). Total dissolved solids (TDS), total suspended
solids (TSS), alkalinity and hardness parameters before and after
coagulation studies were carried out by the process mentioned in
(APHA, 1998).
2.3.2. DGGE analysis of DCM
The denaturing gradient gel electrophoresis (DGGE) was performed to investigate the presence of microbial community in
DCM. DCM was grown for 24 h in nutrient medium at 30 °C. Genomic DNA was isolated from DCM samples and used for polymerase
chain reaction (PCR) amplification of the 16S rRNA genes by using
the forward primer RDB1-GC clamped (F58 CGCCGCCGCGCC
CCGCGCCCGGCCCGCCGCCGCGGCCGCAGTTTGATCCTGGCTCA) and
reverse primer RDB2 (GGACTACCAGGGTATCTAAT). 50 lL composition of reaction mixture was containing 1 pM 1X PCR buffer, 1 nM
of dNTPs, 1 unit Taq DNA polymerase, 2 mM MgSO4, 2 lL of template DNA and 0.25 pM of forward and reverse primers. The PCR
amplifications were carried as initial denaturation at 95 °C for
5 min, 35 cycles of 95 °C for 15 s, 50 °C for 15 s, and 72 °C for
15 s, followed by 10 min final extension at 72 °C. 1% agarose gel
was used to check the purity of amplified PCR product. The DGGE
analysis was performed with Decode Universal Mutation Detection
System (Bio Rad) to obtain concentrated product. Samples were
loaded onto 8% (w/v) polyacrylamide gels (37.5:1, acrylamide: bisacrylamide) in 1X TAE buffer with a denaturing gradient ranging
from 35% to 70% denaturant run at 60 °C, 80 V for 14 h. Gel was
2.3. Decolorization of dye sludge generated under SSF
2.3.1. Isolation of microbial consortium for coagulated dye
decolorization
One gram of soil from highly polluted textile industry sites (Ichalkaranji, India) was inoculated in 250 mL Erlenmeyer flasks containing 10 mL of coagulated dye sludge. 20 mL nutrient broth
(g L1; peptone 10, sodium chloride 10 and beef extract 2) was
A.A. Kadam et al. / Bioresource Technology 176 (2015) 38–46
Table 1
Coagulation study of textile dyestuffs using ZnCl2 as coagulant.
1
2
3
Dyestuffs
% Dye removal
Various textile dyes
Reactive Red 120
Reactive Blue 19
Rubin GFL
Navy blue HE2R
Remazol Red
Remazol Black B
Red HE3B
Orange 122
Navy Blue 2R
Blue 2RNL
Direct Red 5B
Golden Yellow HE2R
Green HE4B
MTD*
TW*
99 ± 0.53
99 ± 0.14
98 ± 0.57
97 ± 0.50
96 ± 0.88
96 ± 0.81
97 ± 1.15
96 ± 0.88
97 ± 1.20
96 ± 0.88
97 ± 0.78
96 ± 0.89
95 ± 1.20
96 ± 1.50
98 ± 0.88
MTD – mixture of textile dyes.
TW – textile wastewater.
Values are mean of three experiments ± SEM.
*
% ADMI removal.
stained with silver stain and visualized (Joshi et al., 2013; Kadam
et al., 2013b).
2.3.3. Decolorization of coagulated dye sludge under SSF
Coagulated dye sludge was produced in the coagulation process.
10 mL of such dye sludge produced from 100 mL of RR120, Reactive Blue 19, Rubin GFL, Navy Blue HE2R, Remazol Red, Remazol
Black B, Red HE3B, Orange 122, Navy Blue 2R, Blue 2RNL, Direct
Red 5B and Golden Yellow HE2R and Green HE4B having concentration 100 mg/L was taken in a 250 mL Erlenmeyer flask. 5 g of
rice bran was added. The pH content was adjusted to 7 using
0.1 N HCl. These test flasks were sterilized and moisture content
was adjusted using sterile distilled water. After adjustment of
moisture content the flask were inoculated with 3 mL of DCM
and incubated at 30 °C at static conditions for decolorization. Similarly abiotic flasks were incubated. All experiments were carried
out in triplicates. In order to measure the decolorization of dyes,
control abiotic flasks and test flasks were extracted with 50 mL
of dimethyl sulfoxide (DMSO) under shaking condition (120 rpm)
for 30 min. The obtained solutions were filtered through the Whatmann filter paper No. 42. Then, it was centrifuged at 7000 rpm for
10 min. The clear supernatant was used for color measurement.
The intensity of color was measured at maximum absorbance
wavelength of respective individual dyes using Hitachi U-2800
spectrophotometer. Percent decolorization was calculated as
reported by Kadam et al. (2013a). Percent ADMI (American Dye
Manufacturing Institute) removal values were used in order to
measure the decolorization of MTD and TW. The color characteristics of MTD and TW are highly erratic in both, concentration and
hues. Spectrum obtained for MTD and TW did not show well
defined peaks in the visible region mainly due to complex mixture
of dyes, and therefore decolorization of the MTD and TW was measured in terms of ADMI value. The American Dye Manufacturers’
Institute (ADMI 3WL) tristimulus filter method was used to measure decolorization of mixture of dyes and textile effluent (Lade
et al., 2012). The ADMI removal ratio was calculated as reported
by Lade et al. (2012). In order to optimize the pH for decolorization
of dye adsorbed substrates by microorganism, the pH of the medium was adjusted to 2, 4, 6, 8 and 10. The flasks were then inoculated with microorganism and incubated at 30 °C at static
condition. The effect of temperature on decolorization was studied
at 10, 20, 30, 40, and 50 °C at static condition keeping the pH 6.5–7.
Moisture content of the medium was adjusted to 75, 80, 85, 90 and
95% in order to study its effect on the decolorization. pH was
41
adjusted to 6–7 and incubated at 30 °C in static conditions after
adjustment of moisture content. 2, 4, 6, 8 and 10 mL dye sludge
of RR120 and TW was taken for studying effect of increasing dye
concentration on decolorization. Decolorization of dye at different
concentration by DCM under SSF was measured after the 24 h of
incubation at static conditions.
2.4. Scale up studies for coagulation and decolorization process
2.4.1. Pilot scale coagulation reactor (CR)
After optimization, coagulation process was scale up by using
the CR. The reactor was constructed by using tank D and K made
up of polypropylene carboys having capacity of 50 L purchased
from (Tarsons product private limited, India). Effluent addition
(A), Coagulant addition (B) and NaOH addition (C) ports were present at the top opening end of tank D (see Supplementary material,
Fig. S1). 50 L of effluent was taken in tank D. The coagulant ZnCl2
(11 mM/L) was added and pH was adjusted to 10 by 0.5 N NaOH
in the tank D. This mixture was agitated at 60 rpm for 10 min by
the motor G connected with stirrer E (see Supplementary material,
Fig. S1). Then it was allowed to stand for 15 min. The dye coagulate
settled at the bottom of reactor was removed by port I from which
coagulate was collected in coagulation collection tank J (see Supplementary material, Fig. S1). The retained solution of dye was
passed in tank K. The final pH adjustment and removal of suspended particle (if any) was done in tank K (see Supplementary
material, Fig. S1). Effluent removed from this reactor was sent outside as treated effluent. Similar 10 rounds of reactor cycles process
was carried out. 500 L of effluent treated in the pilot scale coagulation reactor study. Collected dye sludge from 500 L effluent further
treated in composting bioreactor.
2.4.2. Pilot scale composting bioreactor (CB) for decolorization of
sludge generated in CR
Coagulate generated in coagulation reactor was decolorized in
the CB. The CB was made up of water proof thermocol box having
the dimensions of 48 48 cm square shaped, 15 cm height with
5 cm width. The biomass layer mainly consists of 10 cm in the
reactor. 10 L of dye coagulate obtained from 500 L of TW was
added with the 10 kg of rice bran. The pH and moisture content
of the slurry was adjusted to 7 and 90%. The inoculums of developed microbial consortium (4 L) and 500 mL of active compost
slurry were added in the reactor. The reactor was incubated in
non-sterile conditions and open place. Decolorization was monitored by the method mentioned earlier.
2.5. Phytotoxicity study
Dyes were still present in the environment after removal from
the dye solution by coagulation process. Hence, it is important to
carry out its toxicity studies. Phytotoxicity tests were performed
in order to study the toxic effects of TW, coagulated sludge of
TW, coagulation treated effluent and extracted products from
decolorized coagulated sludge under SSF. Toxicity analysis was
done using Sorghum vulgare and Phaseolus mungo as model plants.
The experiment was carried out at room temperature (10 seeds of
each) by watering separately 5 mL sample per day. Control set was
carried out using distilled water. Lengths of radicles (root) and
plumules (shoot) were recorded on 12th day.
2.6. Statistical analysis
One-way analysis of variance (ANOVA) was performed for the
statistical analysis of data with Tukey–Kramer multiple comparison test using the software Graph Pad InStat version 3.06.
42
A.A. Kadam et al. / Bioresource Technology 176 (2015) 38–46
Table 2
Environmental parameter analysis.
Parameter
% Removal
BOD
COD
TOC
TDS
TSS
MTD
TW
79 ± 0.88
91 ± 0.81
90 ± 0.57
82 ± 1.50
82 ± 1.15
83 ± 0.74
92 ± 1.22
93 ± 0.75
85 ± 1.38
85 ± 1.38
MTD – mixture of textile dyes.
TW – textile wastewater.
Values are mean of three experiments ± SEM.
3. Result and discussions
3.1. Coagulation studies
3.1.1. Coagulation of dyes by ZnCl2
Chemical coagulation is addition of the chemicals which leads
to alter the physical state of suspended as well as dissolved solids
and their removal by means of sedimentation (Verma et al., 2012).
1
2
3
4
5
Coagulation is considered as the most successful treatment
because of efficacy of the process and immediate treatment of
TW (Huang et al., 2009). It was extremely necessary if considered
large volume of TW generated daily from textile industries
(Kadam et al., 2013b). Various textile dyestuffs at the concentration of 100 mg/L such as RR120, Reactive Blue 19, Rubin GFL, Navy
Blue HE2R, Remazol Red, Remazol Black B, Red HE3B, Orange 122,
Navy Blue 2R, Blue 2RNL, Direct Red 5B, Golden Yellow HE2R and
Green HE4B showed 99, 99, 98, 97, 96, 96, 97, 96, 97, 96, 97, 96
and 95% removal during coagulation by ZnCl2, respectively
(Table 1). 96% and 98% ADMI removal was obtained for MTD and
TW, respectively (Table 1). The effectiveness of coagulation process
could be enhanced by adequate selection of coagulant, flocculent
aids, and optimization of process parameters such as pH, mixing
time, temperature and coagulant concentration (Tan et al., 2000).
The optimum pH for coagulation of RR120 and TW was found to
be 10 and no dye removal was observed at acidic pH (Fig. 1[1]).
While coagulation of dyes observed at pH 12 was found to be consistent as that of pH 10. The agitation time of 1 min showed similar
coagulation of dye as that of the agitation time of 2, 3, 4 and 5 min
(Fig. 1[2]). Requirement of minimum agitation reduce the cost of
treatment. Coagulation of dye was increased with an increase in
concentration of the coagulant. 11.0 mM/L concentration was
found to be optimum (Fig. 1[3]). The coagulation reaction was
found to be temperature independent (Fig. 1[4]). These optimized
parameters might help during the scale up of the process. Broad
temperature range of the process suggests its applicability at versatile environmental conditions. Most of the earlier coagulation
studies were carried out using single dye (Verma et al., 2012).
Coagulation of structurally versatile dyes, MTD and TW was inadequate to judge the performance of particular coagulant. This
result signified the use of ZnCl2 as a coagulant for removal of various textile dyes, MTD and TW. Additionally, as far as our knowledge this is the first report suggesting ZnCl2 as coagulant for
textile wastewater treatment. Cost of coagulant ZnCl2 used has
influences the overall treatment cost of the process. Therefore, cost
evaluation carried out at one of the retail and wholesale trade company Alibaba.com. It has been showed that the market prize of
ZnCl2 (US $500–1000/Metric Ton), FeCl3 (US $500–600/Metric
Ton) and AlCl3 (US $1000–2000/Metric Ton). Hence, from this analysis it was clear that market cost of ZnCl2 has been comparable
with the costs of conventionally used coagulants such as FeCl3
and AlCl3. Secondary dye sludge was major drawbacks of the
Table 3
Decolorization of coagulated dye by developed microbial consortium under SSF.
Sr. no
Dyestuffs
% Decolorization in 72 h
1
Various textile dyes
Reactive Red 120
Reactive Blue 19
Rubin GFL
Navy blue HE2R
Remazol Red
Remazol Black B
Red HE3B
Orange 122
Navy Blue 2R
Blue 2RNL
Direct Red 5B
Golden yellow HE2R
Green HE4B
MTD*
TW*
96 ± 2.33
97 ± 0.66
98 ± 0.57
96 ± 1.20
94 ± 2.08
93 ± 1.55
93 ± 1.13
92 ± 1.45
95 ± 1.55
93 ± 1.55
95 ± 1.43
85 ± 2.64
92 ± 1.43
92 ± 1.54
98 ± 0.75
6
7
8
9
2
3
10
Fig. 2. DGGE analysis of DCM.
MTD – Mixture of textile dyes.
TW – textile wastewater.
Values are mean of three experiments ± SEM.
*
% ADMI removal.
43
A.A. Kadam et al. / Bioresource Technology 176 (2015) 38–46
100
100
[1]
[2]
80
% Decolorization
% Decolorization
80
60
40
20
60
40
20
0
0
2
4
6
8
10
10
20
30
40
50
8
10
Temperature (oC)
pH
120
100
[4]
[3]
100
% Decolorization
% Decolorization
80
80
60
40
60
40
20
20
0
0
75
80
85
90
95
2
4
6
Dye coagulate (mL)
Moisture content (%)
% Decolorization of Coagulated RR-120
% ADMI removal of coagulated textile effluent
Fig. 3. Optimization of decolorization parameters such as [1] pH, [2] temperature, [3] moisture content and [4] dye coagulate concentration.
coagulation technique (Chu, 2001). Very less emphasize was given
to its further treatment (Sanghi et al., 2006). Therefore coagulated
dye sludge was decolorized under the solid state fermentation conditions in further study.
3.1.2. Investigation of coagulation mechanism by ZnCl2
The visible spectrum obtained from the samples in which dye
sludge acidified to neutral pH suggests similar shape of the spectrum compared to the untreated RR120 (see Supplementary material, Fig. S2). While, slight decrease in absorbance in treated
samples was observed due to the residual loss of color during the
process. These results indicate that the structure of the dye was
remained intact during the coagulation process. In presence of
NaOH and ZnCl2 it forms the Zn(OH)2 as the precipitate as shown
in Eq. (1).
ZnCl2 þ NaOH ! ZnðOHÞ2 ðPrecipitateÞ þ NaCl
ð1Þ
Therefore, it was proposed that RR120 has adsorbed on Zn(OH)2
precipitate and get removed from the solution without altering its
original structure. FTIR analysis of coagulate without RR120 and
coagulate with RR120 showed the different spectrum profile that
suggests the surface modification of the sludge properties after
dye removal from the solution (see Supplementary material,
Fig. S3). ZnCl2 coagulate without RR120 and with RR120 represents
the peaks such as 3435 and 3435 cm1 for OH stretching, 464 and
474 cm1 corresponds to the Zn–O stretching (Aboulaich et al.,
2012) confirms formation of Zn(OH)2 precipitate. At the same time,
ZnCl2 coagulate with RR120 showed presence of additional
absorption peaks such as 1510, 1378, 1344, 1281, 1189 and
1124 cm1 which represents NH3 deformation, C–H deformation,
S–O stretching, C–N vibrations, S@O stretching and C–OH stretching, respectively (see Supplementary material, Fig. S3), due to
RR120 adsorption. This results indicate that coagulate Zn(OH)2
formed during coagulation process adsorbs the textile dyes
RR120 and removes it from the solution. FE-SEM analysis (see
Supplementary material, Fig. S4(a and c)) showed different surface
morphologies for coagulate without and with RR120, might be due
to deposition of the dyes by adsorption process on Zn(OH)2. For
further understanding of mechanism FE-SEM EDX analysis was
carried out (see Supplementary material, Fig. S4(b and d)) The elemental composition coagulate without dye suggests the presence
of Zn and O that confirmed the formation of precipitate of Zn(OH)2,
but ZnCl2 coagulate with RR120 showed presence of 6.76% carbon
indicates adsorption of organic dye RR120 on Zn(OH)2 (see
Supplementary material, Table S1). Similarly, SEM and FTIR
characterization of coagulants polyferric sulphate (PFS) and polyacrylamide (PAA) was observed by Moussas and Zouboulis, 2009.
FE-TEM analysis results elucidate the different surface morphology
by coagulates with and without RR120 (see Supplementary material, Fig. S5). Coagulate without dye showed fairly crystallized
structure while coagulate with RR120 had well crystallized pseudo
cubic structures (SM 4). Similar structure of coagulates of MnCl2
and MgCl2 was observed earlier (Bouyakouba et al., 2011). Hence,
mechanism of color removal by ZnCl2 is probably involves: all
the zinc ions from solution were converted into zinc hydroxide
precipitate (Zn(OH)2) at pH greater than 10; and the precipitate
44
A.A. Kadam et al. / Bioresource Technology 176 (2015) 38–46
structure provides a large adsorptive surface area having a positive
electrostatic surface charge. Similar investigation of possible coagulation mechanism of MgCl2 was also proposed by Gao et al., 2007.
Therefore, coagulation mechanism of ZnCl2 for removal of the dyes
was proposed to be charge neutralization and an adsorptive
coagulating mechanism using the above mentioned different techniques such as UV–vis spectrophotometry, FTIR, FE-SEM EDX and
FE-TEM.
3.1.3. TOC, COD, BOD, TSS and TDS reduction analysis
Textile effluent released from industries has very high TOC,
COD, BOD, TSS and TDS values (Saratale et al., 2011). It is an extremely essential part of the any wastewater treatment to reduce
these values significantly as being an efficient treatment
(El-Gohary and Tawfik, 2009). The solution obtained after coagulation of dyes from TW by ZnCl2 showed 90, 91, 79, 82 and 82%
reduction of TOC, COD, BOD, TSS and TDS, respectively (Table 2).
Similarly 93, 92, 83, 85 and 85% reduction of TOC, COD, BOD, TSS
and TDS was found for the solution obtained after the coagulation
of dyes from MTD (Table 2). Significant reduction in environmental
parameters for TW and MTD suggests an efficient treatment for TW
removal in addition to the color removal. These experiments conclude that ZnCl2 is an efficient coagulant having excellent color
removal as well as TOC, COD, BOD, TSS and TDS reduction
capacities.
3.2. Decolorization of coagulated dyestuffs under solid state
fermentation
The isolated DCM was analyzed by DGGE technique in order to
investigate the number and composition of bacterial community
present. In the DGGE analysis PCR amplified DNA using 16 S rRNA
primers were separated on denaturant gradient gel. Hence, each
band obtained on gel indicates the presence of an individual bacterium. Therefore, the consortium mainly contains total of 10 distinctive bands suggests the presence of 10 different bacteria’s
(Fig. 2). Generation of sludge is the major limitation of the coagulation process (Chu, 2001). Hence, the decolorization of sludge generated under the solid state fermentation provides novel approach
for textile dyes decolorization, because coagulation is considered
as most widely used technique for TW treatment. Therefore, the
decolorization of coagulated dyes (dye sludge) under solid state
fermentation is probably a prominent technology for dye
decolorization. In this study dyes sludge of RR120, Reactive Blue
19, Rubin GFL, Navy Blue HE2R, Remazol Red, Remazol Black B,
Red HE3B, Orange 122, Navy Blue 2R, Blue 2RNL, Direct Red 5B,
Golden Yellow HE2R and Green HE4B showed 96, 97, 98, 96, 94,
93, 93, 92, 95, 93, 95 and 85% decolorization by DCM in 72 h,
respectively (Table 3). Similarly, 92 and 98% of ADMI removal for
textile dyes mixture and TW was observed by DCM in 72 h, respectively (Table 3). Various parameters such as pH, temperature,
moisture content and dye coagulate concentration were studied
for the decolorization of dye coagulate of RR120 and TW. The pH
of medium was adequate parameter for decolorization by bacteria
in the context of dye uptake and its enzymatic transformation and
degradation. The optimum pH and temperature for decolorization
of dye sludge Reactive Red 120 and textile effluent was found to be
8 and 30 °C, respectively (Fig. 3[1]). The increase in temperature
causes an increase in moisture loss and that resulted in decrease
in the decolorization percentage (Kadam et al., 2014). The moisture
content 90% was found to be optimum for decolorization
(Fig. 3[2]). The decolorization percentage was decreased below
90% moisture content might be due to limited growth of the microorganisms and less accessibility of microorganisms to dye molecules (Kadam et al., 2014). The decolorization was decreased
with an increase in dye sludge concentration (Fig. 3[3], [4]).
Increased dye concentration might have some toxic effect on the
dye decolorizing microorganisms (Saratale et al., 2011). This result
suggests that successful decolorization of coagulated dyestuffs
under solid state fermentation conditions as a novel hybrid treatment approach for textile dyes.
3.3. Scale up studies
3.3.1. Scale up of coagulation process using pilot scale coagulation
reactor (CR)
Initially coagulation study was carried out using batch scale.
However, the scale up was carried out using the CR. The 50 L of
TW was treated in CR for one cycle. Average 94% ADMI removal
was observed from highly concentrated TW for 10 successive
cycles. Hence, total of 500 L of TW was treated in 10 cycles of CR.
This results obtained was found to be extremely significant considering the volume of wastewater and scale up of the process. Scale
up of process at pilot level was essential in order to access applicability of process at large scale. Coagulate generated in the CR was
collected in the coagulate collection tank. The retained dye
100
% ADMI removal at surface of the reactor
90
% ADMI removal at bottom of the reactor
80
% Decolorization
70
60
50
40
30
20
10
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
Number of days
Fig. 4. Decolorization of coagulated dye in the surface as well as bottom of composting bioreactor (CB).
45
A.A. Kadam et al. / Bioresource Technology 176 (2015) 38–46
Table 4
Phytotoxicity analysis of TW before and after treatment by using Phaseolus mungo and Sorghum vulgare.
Parameters
Germination
(%)
Plumule
(cm)
Radicle (cm)
Phaseolus mungo
Phaseolus mungo
Water
TW
Dye sludge
after
coagulation
Coagulation
treated
effluent
Extracted
products from
SSF biomass
Water
TW
Dye sludge
after
coagulation
Coagulation
treated
effluent
Extracted
metabolites from
SSF biomass
100
30
40
90
80
100
40
40
90
80
12.0 ± 0.5
5.2 ± 0.1⁄⁄
6.3 ± 0.2⁄⁄
11.0 ± 0.4⁄
10.0 ± 0.3⁄
9.2 ± 0.2⁄
4.0 ± 0.2⁄⁄
4.5 ± 0.6⁄⁄
8.5 ± 0.4⁄
8.0 ± 0.2⁄
8.3 ± 0.3
5.0 ± 0.4⁄⁄
5.3 ± 0.5⁄⁄
7.0 ± 0.1⁄
6.5 ± 0.6⁄
8.1 ± 0.2⁄
4.7 ± 0.1⁄⁄
4.1 ± 0.5⁄⁄
7.5 ± 0.3⁄
6.5 ± 0.4⁄
The values are significantly different from control at P < 0.01,
three experiments ± SEM.
⁄
⁄⁄
P < 0.001 by one-way analysis of variance (ANOVA) with Tukey–Kramer comparison test. Values are mean of
solution then passed to treat in effluent collection tank for its final
pH adjustments and removal of residual particles. Most of earlier
coagulation studies were carried out by using the single model
dye (Verma et al., 2012). TW treatment and scale up of studies of
coagulation process suggested its wide use for the treatment of
the effluent of textile industry (El-Gohary and Tawfik, 2009).
3.3.2. Scale up of SSF process using pilot scale composting bioreactor
(CB)
In the scale up of the coagulation process, collected dye sludge
from 500 L effluent treated in 10 cycles was decolorized under the
CB. Composting of hazardous materials contaminated soils is an
emerging ex situ bio-treatment technology (Antizar-ladislao
et al., 2004). However, effective biodegradation at the laboratory
and/or field scales using the composting process of PAHs (Canet
et al., 2001), explosives (Gunderson et al., 1997), petroleum hydrocarbons (Jørgensen et al., 2000; Namkoong et al., 2002) and chlorophenols (Laine and Jørgensen 1997) were reported earlier.
Composting–bioremediation using various composting systems
such as enclosed systems as tunnels and modular containers or
open-air systems includes static aerated (pilesil) and mechanically
turned (windrow) (Potter et al., 1999; Jørgensen et al., 2000; Sasek
et al., 2003). Therefore, composting of dye sludge ultimately
enhances the effectiveness of coagulation process by degrading/
detoxifying generated sludge. The decolorization was studied at
the surface as well as bottom of the reactor. The samples were
taken from 5 different locations i.e. 5 cm inside from each corner
and 1 sample from center of bioreactor. (Supplementary material,
Fig. S6) showed the decolorization after 5, 10, 20 and 30 days. Significant decolorization was observed in composting for one month
under non sterile conditions and simply as dumping bioreactor
(see Supplementary material, Fig. S6). Decolorization was higher
at the surface of reactor when compared with the bottom. This
effect might be due to the initial dominance of aerobic microorganisms and simultaneously slow development of anaerobic microorganisms (Fig. 4). Decolorization at the bottom as well as surface
became same after 20 days. This might be due to the development
of anaerobic micro flora. 92% ADMI removal was found after
twenty days (Fig. 4) which further increased to 99% ADMI removal
after 30 days. This result suggests low cost, eco-friendly composting technology for bioremediation of highly concentrated dye
sludge coupled with popular chemical coagulation method of textile dyestuffs.
3.4. Phytotoxicity analysis
Textile dyes were toxic in nature for plants (Paul et al., 2012).
After the biodegradation of dyes, toxicity of textile dye towards
the plants was decreased significantly (Kadam et al., 2014). Phaseolus mungo and Sorghum vulgare were reported to be model plants
for phytotoxicity studies (Paul et al., 2012). During coagulation
process textile dyes were removed from effluent but still present
in the sludge. Therefore, it was essential to check toxicity effect
of the dye-sludge. The phytotoxicity studies for TW, coagulated
sludge of TW, coagulation treated effluent and extracted products
from decolorized coagulated sludge under SSF reveals that the germination percentage in presence of TW and coagulated sludge of
TW were decreased significantly when compared to the control.
The germination percentage of coagulation treated effluent and
extracted product from SSF biomass were similar to water
(Table 4). The results explain the decrease in toxicity of coagulation
generated sludge under SSF. In the similar context, plumule and
radicle lengths of both the model plants reduced significantly in
presence of TW and coagulated sludge of TW when compared with
control (Table 4). However no effect was observed in case of SSF
treated coagulated sludge. Although the coagulation is an efficient
process, the dye sludge produced was found to toxic in nature for
plants. But, significantly reduction in the phytotoxicity was
observed after the SSF treatment for dye sludge. Therefore, this
result indicates that the reduction of dyes toxicity completely after
the coagulation process along with biodegradation under the SSF.
4. Conclusions
Coagulant ZnCl2 was utilized for removal of textile dyes. Charge
neutralization and adsorption was found to be dye coagulation
mechanism of ZnCl2. Significant reduction in TOC, COD, BOD, TSS
and TDS of MTD and TW after ZnCl2 treatment demonstrated its
high efficiency as coagulant. One of the major drawbacks of coagulation process has resolved by decolorization of generated sludge
under low cost and eco-friendly SSF. Hybrid technique combining
coagulation and SSF was ultimately enhances effectiveness of coagulation process. Batch scale and pilot scale experiments of coagulation and SSF for removal of dyes suggests ecofriendly, low cost and
efficient treatment approach.
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.2014.10.
137.
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