Journal of Cleaner Production 161 (2017) 560e566

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Journal of Cleaner Production

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An eco-friendly approach towards treatment of tannery lime liquor

using halo tolerant alkaliphile of extreme origin
Yasmin Khambhaty a, b, *, Khyati Ojha b, R. Akshaya b, E. Madhava Charyulu c
a
Leather Process Technology Division, CSIR-Central Leather Research Institute, Adyar, Chennai, 600 020, Tamil Nadu, India
b
Biological Materials Division, CSIR-Central Leather Research Institute, Adyar, Chennai, 600 020, Tamil Nadu, India
c
Centre for Biotechnology, Anna University, Chennai, 600 025, India

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

Article history: The management of tannery lime liquor using halo tolerant bacterium of extreme origin (accession
Received 3 January 2017 number MTCC 5899) is reported in the present study. The isolate is capable to bring simultaneous
Received in revised form reduction in pH as well as Total Dissolved Solids (TDS) of highly alkaline lime liquor (~13.6 pH) within a
3 April 2017
reasonable time period. The strain is found to be growing at a wide range of pH (9.0e13.6) and salt
Accepted 27 May 2017
Available online 29 May 2017
concentration of 0e10%. Factors like amount of inoculum, dilution of liquor, type and concentration of
carbon and nitrogen source used and initial TDS levels were observed to have a profound effect on the
neutralization and reduction in TDS levels of lime liquor. A reduction in BOD5 and COD by ~62% and 65%
Keywords:
Biological neutralization
respectively was observed when the liquor was added with suitable carbon source. The present study
Enrichment was targeted to exploit the ability of alkaliphilic bacteria to reduce pH in order to provide an economic
Lime liquor process for the nullification of highly alkaline lime liquor along with reduction in TDS, eventually making
Micrococcus sp. the liquor environmentally safe for discharge. This study paves way to greener route towards the
Total Dissolved Solids treatment of tannery lime liquor, for the first time using halo alkaliphilic Micrococcus sp.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction however, other processing units viz., soaking, liming-deliming,

The discharge of wastewaters, containing hazardous com-
pounds into natural bodies adds substantially to the accumulation
of recalcitrant pollutants (Kumar et al., 2005). The leather industry
has been classified as one of the highly polluting industries and
there is concern that leather making activity can have adverse
impact on the environment (Kanagaraj et al., 2006). Hence, despite
of the industry making traceable and visible impacts on socio-
economics through employment generation and export earnings,
the industry has gained negative image in the society owing to the
resulting pollution (Thanikaivelan et al., 2004).
The leather making process can be split into four main cate-
gories: i) storage and beam house operations ii) tan yard operations
iii) post-tanning operations and iv) finishing operations (Durai and
Rajasimman, 2011). The use of sodium chloride during raw material
preservation is a major contributor of TDS in tannery wastewater,
* Corresponding author. Leather Process Technology Division, CSIR-Central
Leather Research Institute, Adyar, Chennai, 600 020, Tamil Nadu, India.
E-mail address: yasmink@clri.res.in (Y. Khambhaty).
bating and degreasing also contribute to the TDS load as each
unit discharges effluent containing inorganic components like
sulphates, sulphides, carbonates, bicarbonates and calcium (Kumar
et al., 2005). The TDS of soak liquor varies from 33,000 to
40,500 ppm while that of lime liquor varies from 38,500 to
45,500 ppm (Pachpande and Ingle, 2004). The use of high quanti-
ties of lime and sodium sulphide creates an extremely alkaline
environment resulting in the pulping of hair and its subsequent
removal. The disadvantages include contribution of 70e80% of total
Chemical Oxygen Demand (COD) of effluent, sulphide being highly
toxic with obnoxious odor when left untreated causes major
problems in the sewer and poses health hazards due to severe
alkaline conditions (Sanghi and Singh, 2012). Among the problems
in complying with standards, high TDS levels in the tannery
effluent is yet to be resolved. While the treated tannery effluent has
TDS value of about 7000e10,000 ppm, the Indian standard for the
same is prescribed to be 2100 ppm (Sivakumar et al., 2005).
Although there are well established Common Effluent Treatment
Plant (CETP's) for the management of effluents from various tan-
ning operations, the lime liquor needs special attention since the
available chemical treatments are not very effective and add

http://dx.doi.org/10.1016/j.jclepro.2017.05.167
0959-6526/© 2017 Elsevier Ltd. All rights reserved.
 Y. Khambhaty et al. / Journal of Cleaner Production 161 (2017) 560e566
substantially to the pollution load. Systematic studies on the world
wide treatment technologies and advances for pollution prevention
from tannery chemicals and wastewater have been undertaken
(Lofrano et al., 2013; Kowalik -Klimczak and Gierycz, 2014). Several
methods such as membrane separation (Alves and de Pinho, 2000;
Viero et al., 2002), reverse-osmosis (Cassano et al., 2001), electro-
dialysis (Raghava Rao et al., 1989) etc., have been employed for
the treatment of tannery effluent. However, these methods pose
problems like rapid scaling and befouling of the membrane
resulting in reduced flux rate and performance, frequent mem-
brane cleaning and replacement may therefore be necessary, which
would also enhance the maintenance and operating cost of the
technology (Ranganathan and Kabadgi, 2011; Shreesadh et al.,
2013; Yusif et al., 2016). Therefore, the most sustainable solution
of TDS reduction is to adopt process innovations (Sivakumar et al.,
2005).
Total Dissolved Solids provide information on the amount of
both organic and inorganic compounds which may in many cases
remain persistent and result in a cumulative toxic effect (Genschow
et al., 1996). The variations in the amounts of Dissolved Solids can
be detrimental since the density of TDS determines the flow of
water in and out of an aquatic organism's cell. High concentration
of TDS may reduce the water clarity leading to a decrease in
photosynthesis and when added with toxic compounds/heavy
metals, leads to increase in temperature. This can often be harmful
to many aquatic forms (Kumar et al., 2005).
In a recent study, liming and pickling streams were mixed at
different ratios with a view to develop a simple and economically
viable method for tannery effluent treatment. This resulted in
effluent with pH very close to neutral and caused a remarkable
reduction in COD (88%), BOD (94%) and TS (97%) when the lime:
pickle ratio was kept at 1: 4 (Mottalib et al., 2014).
One of the major obstacles in treating the lime liquor through
biological means is its high alkaline pH (12.7e13.4) (Pachpande and
Ingle, 2004) as terrestrial microbes cannot grow/survive in such
harsh condition. One ideal option to treat such effluents would be
the use of alkaliphiles which would not only survive but grow
luxuriously in such environment. Certain group of microorganisms
demonstrates growth in highly alkaline conditions and in turn re-
duces the pH of the medium through its biochemical pathways.
Reports suggest that more than 3e4 units of reduction in pH were
observed by group of halophiles and alkaliphiles. Hence, making
use of those species to reduce the pH of the lime liquor by natural
means is found to be attractive and once the pH is reduced, the
effluent may be treated in CETP along with other sectional streams.
The biological treatment of alkaline effluent generated from
paper and pulp (Kumar et al., 2005a), textile (Kumar et al., 2005b;
Kumar and Kumar, 2006), beverage (Kumar and Kumar, 2006a;
Kulshreshtha et al., 2010) and chlor-alkali industries (Jain et al.,
2011), have established the prospective use of alkaliphiles for
bioremediation which further needs to be proved for lime liquor of
leather industry. The ability of extremophilic bacteria growing at
high pH could be exploited for treatment of this type of wastewater.
In the present study, an attempt is made to address two major
concerns of the lime liquor viz., exceedingly alkaline pH and
elevated levels of TDS. A process was established wherein; the
ability of extreme alkaliphilic bacteria to cause changes in pH even
in a well buffered, carbohydrate containing medium and require-
ment of Naþ for their growth was used to neutralize the liquor. To
the best knowledge of the investigators there are no reports on
simultaneous reduction of pH and TDS using bacteria with special
emphasis on lime liquor. Management of lime liquor through bio-
logical means will certainly reduce the pollutant load to CETP in
addition to making the process more environmental friendly.
561
2. Materials and methods
2.1. Sample collection and isolation of alkaliphiles
Lime liquor and sludge were collected from pilot plant of CSIR-
CLRI, Chennai, India. Bacteria were isolated immediately after
collection of sample on modified Horikoshi medium (Horikoshi,
1999) containing g/L of glucose 10, peptone 5, yeast extract 5,
K2HPO4 1, MgSO4.7H2O 0.2 and agar 20, pH was adjusted to 10.0
using a saturated solution of NaOH. Isolation of bacteria was done
by conventional methods of serial dilution (Yapici et al., 2008). For
sludge samples nearly 1.0 g of sludge was suspended in 100 mL
sterile saline solution, mixed thoroughly and subsequently sub-
jected to serial dilution. 200 mL from each dilution was plated in
triplicates on medium described above and incubated at 35 ± 2 οC
for 24 h. The isolated bacteria were purified by repeated sub
culturing on the same medium and preserved on slants at 4 οC.
2.2. Studies on pH and salt tolerance and neutralization of modified
Horikoshi medium
The distinctly different bacteria obtained as a result of screening
experiment were tested for their ability to grow in liquid medium
with different pH viz., 6.0, 8.0, 10.0, 11.5, and 13.5. A different set of
experiment was also conducted to check the salt tolerance ability
by supplementing the liquid medium (pH 10) with sodium chloride
in the range of 2.0e10%. A control was maintained which did not
contain any salt added. After inoculation, the tubes were monitored
visually for the growth of bacteria for 24 h.
The cultures screened above were subjected to neutralization of
modified Horikoshi broth (now on referred as growth medium) and
dispensed in 100 mL of this medium into different flasks and
autoclaved at 121  C for 15 min. After autoclaving, pH was adjusted
to 10.0, 12.0 and 13.6 with separately autoclaved saturated solution
of NaOH. Inoculum was prepared by adding a loopful of the selected
cultures into the growth medium, incubating at 35 ± 2  C at
150 rpm and allowed to grow for 16e18 h (till it reached an OD of
~2.0, approx.1.8  104e1.8  106 cells/mL). This was then added at a
ratio of 100:10 (Medium: Inoculum) to the growth mediums with
different pH. Reduction in pH was monitored regularly.
2.3. Neutralization of tannery lime liquor
The potential isolate obtained, was exploited for neutralization
of lime liquor (pH ~13.6) collected from pilot plant of CSIR-CLRI,
Chennai. This liquor was enriched with 1.0% glucose and 0.5%
peptone (separately autoclaved) and added to the sterilized lime
liquor in aseptic conditions. Inoculum was prepared as mentioned
in previous section and added into flasks containing 100 mL of
enriched liquors. A control flask with enriched lime liquor but
devoid of inoculum was also maintained. These were incubated at
35 ± 2  C at 150 rpm and reduction in pH and TDS was monitored at
regular time intervals. The experiment was repeated three times
independently each with three replicates. The pH was monitored
with pH meter (Eutech, Thermo Scientific) and TDS determined
according to standard method (APHA, 1998).
2.4. Identification of the promising bacterium
Bacterium was identified on the basis of morphological, physi-
ological and biochemical procedures. The identification was further
confirmed by 16S rDNA sequence analysis. Cell morphology was
examined by light microscopy (Nikon E600). Genomic DNA was
isolated from purified culture pellet. Using rDNA sequence specific
consensus primer, ~1.5 kb fragment was amplified using high
562 Y. Khambhaty et al. / Journal of Cleaner Production 161 (2017) 560e566
fidelity PCR Polymerase. The PCR product was cloned and bidirec-
tionally sequenced using forward and reverse primers and one
internal primer. The 16S rDNA gene sequence was compared with
sequences in the nucleotide database by using the BLAST algorithm
at the NCBI site (Weisburg et al., 1991). Sequence has been sub-
mitted to the GenBank database under accession number
KX868097.
2.5. Modified method for inoculum preparation
For experiments conducted so far, inoculum was prepared by
adding a loopful of the selected cultures into the growth medium,
incubated at 35 ± 2  C at 150 rpm and allowed to grow for 16e18 h
(till it reached an OD of ~2.0, approx.1.8  104e1.8  106 cells/mL).
This was preceded by withdrawing required quantity of this inoc-
ulum and adding to the growth medium such that a ratio of 100: 10
(Medium: Inoculum) is maintained. However, with a view to
accelerate the neutralization process, two different methods for
inoculum preparation were adopted. Both the methods followed
similar steps until withdrawal of required quantity of inoculum. For
the first method, this withdrawn inoculum was centrifuged at
8000 rpm at 4  C for 10 min. The pellets were collected under
sterile conditions and directly inoculated into the enriched lime
liquor. Nonetheless, for the second method, similar step were fol-
lowed till collection of pellet. However, here, the collected pellet
was then suspended into required quantity of sterile freshly pre-
pared growth medium and agitated well to make slurry, which was
eventually used as inoculum. pH and TDS was monitored at regular
intervals.
2.6. Improvisation of various parameters for maximum pH and TDS
reduction
2.6.1. Study on varying dilutions of effluent
Lime liquor to be treated had a pH of ~13.6 and TDS of
~20,000 ppm. This liquor was subjected to dilution with water at
varying ratios; i.e. 75:25 (75 mL lime liquor þ 25 mL water), 50:50
and 25:75. The undiluted lime liquor was kept as control. Hundred
mL of each of these lime liquors were autoclaved and added with
separately sterilized glucose (1%) and peptone (0.5%). Ten mL of cell
slurry was inoculated into each of these enriched lime liquors and
incubated at 37  C at 150 rpm. pH was monitored at regular in-
tervals, however, TDS was monitored only after reduction in pH was
observed.
2.6.2. Study on higher inoculum size
Indiscriminate to any treatment conditions, the inoculum size
was kept constant (100:10). An attempt was made to find the effect
of higher inoculum size on the neutralization process. For this
inoculum was prepared as mentioned in section 2.5 (second
method), wherein 10, 25, 50 and 100 mL of culture was withdrawn
separately and centrifuged at 10,000 rpm. The pellet obtained were
re-suspended in corresponding amount of growth medium and
agitated well to form cell slurry, under aseptic conditions.
Tannery lime liquor to be treated had pH of ~13.6 and TDS of
17,520 ppm. This liquor (100 mL) was autoclaved and enriched with
glucose and peptone in four flasks. Different cell slurry as obtained
earlier were inoculated into the enriched lime liquors and incu-
bated at 37  C at 150 rpm. pH was monitored at regular intervals.
2.6.3. Effect of initial TDS levels over time
In order to identify the role of initial TDS levels, an experiment
was set up where in lime liquors having TDS levels of 12,000 and
36,000 ppm at the start of the experiment were taken. The lime
liquors were enriched with separately sterilized carbon and
nitrogen sources and TDS levels were checked by standard
procedure.
2.6.4. Study on the effect of various types and concentration of
carbon and nitrogen sources
In order to study the effect of different carbon and nitrogen
types used for enrichment, experiments were conducted using
different conventional carbon substrates viz., glucose, sucrose,
galactose, maltose and fructose and nitrogen substrates viz.,
peptone, yeast extract, urea, ammonium nitrate and sodium nitrate.
Fresh inoculum was prepared and ten mL of cell slurry was inoc-
ulated into enriched lime liquors and incubated at 37  C at 150 rpm.
Reduction in pH was noted regularly. After selection of the suitable
carbon and nitrogen source, their concentrations were also
optimized.
However, with a view to make the process economic, the pos-
sibility of using different unconventional cheaper carbon sources
like sugarcane bagasse, wheat bran and jack fruit seed powder was
envisaged. These unconventional substrates were used in combi-
nation in the medium at the concentration ranging from 1 to 3%
(based on sugar content). Inoculum having bacterial density of CFU
1.8  104e1.8  106 cells/mL was used for inoculation. Various
substrates used in the present study were analyzed for C, H, N and S
content (Perkin Elemer, CHNS analyzer-2400, USA).
2.7. Bacterial arbitrated neutralization process and comparison
among conventional and bacterial neutralization
In order to prove bacterial mediated neutralization process,
experiments were conducted with control (no bacteria), in the
presence of heat killed and live bacterial cells. Heat killed cells were
prepared by heating the cell suspension at 80  C for 30 min (Jain
et al., 2011). Control experiment was set to evaluate the shift in
pH due to abiotic factors. A comparative study was also conducted
to ascertain the amount of TDS generated by conventional acid
neutralization (hydrochloric acid, sulfuric acid and nitric acid) and
biological neutralization methods and compared with control
(without any treatment).
2.8. Analysis of liquor before and after biological treatment
The effluent from the dehairing process for control and exper-
imental groups were analyzed for other parameters such as BOD5
and COD (APHA, 1998).
2.9. FTIR analysis
In order to record the FTIR spectra, the bacterial culture broths
were centrifuged at 8000 rpm for 20 min. The culture supernatant
was lyophilized and stored at 20  C. The IR spectra of the samples
were taken using FT-IR spectrum 2000 (Perkin Elmer, USA) using
KBr disc method.
3. Results
3.1. Screening of potential alkaliphiles and studies on pH and salt
tolerance
In an endeavor of exploring alkaliphilic bacteria, isolation was
done to entrap the potential bacterial flora from the specific site.
During the screening experiment, among 12 isolates which were
purified, four isolates were selected for pH and salt tolerance
studies based on their distinctly different morphologies. It was
observed that except YK-I none other strains grew at pH 6.0. Iso-
lates YK-J and YK-K could grow moderately only in the range of pH
 Y. Khambhaty et al. / Journal of Cleaner Production 161 (2017) 560e566 563

8.0e10.0 classifying them as alkaliphiles. However, strain YK-L Table 2

exhibited very good growth even at pH > 10.0, hence classified as Morphological and biochemical characteristics of Micrococcus sp.

strict alkaliphile (Table 1). In case of salt tolerance YK-L exhibited Test parameter Result
very good growth from concentrations ranging from 0 to 6%, Form Cocci
however, some growth was observed even at higher concentra- Gram staining Positive
tions. Similar growth pattern was recorded for YK-I but with a Growth on Mac Conkey agar No growth observed
lesser intensity (Table 1). This ability of alkaliphiles to grow luxu- Spores e
Production of pigment Yellow
riantly even at high pH and salt concentration put together with
Indole production -ve
their capability to reduce pH, emerged them as attractive candi- Nitrate reduction þve
dates for management of alkaline lime liquor. Voges Proskaver -ve
Citrate utilization -ve
Gelatin hydrolysis þve
3.2. Neutralization experiments
Catalase þve
Urease -ve
Isolates YK-I and YK-L were employed to neutralize the growth
medium with different pH. It was observed that YK-L could
neutralize the medium with pH 10.0, 12.0 and 13.6 to 6.5, 7.4 and
number KX868097.
8.9 respectively recording a reduction by 4e5 units within 24 h. At
the same time, YK-I could bring down the pH of all the three sets to
6.9, 9.2 and 10.1 which was almost 3e4 units within similar time of 3.4. Modified method for inoculum preparation
incubation (Fig. S1). Both these isolates had good potential to
reduce the pH of modified growth medium within reasonable time Based on the data of neutralization experiment, it was observed
period. However, YK-L was chosen for further studies and experi- that time taken for pH and TDS reduction was fairly high and hence,
ment was conducted using this isolate and inoculated in lime liquor a modified method for the preparation of inoculum was designed
having an initial pH of 13.5 and TDS of 36,000 ppm. Reduction in pH before optimizing other parameters. Accordingly, the flask in which
was noticed by almost 20% within 24 h, representating a reduction pellets were added directly, exhibited a reduction in pH by almost
by approximately 2.5 units which was almost half than that for 28% which was comparatively better than the results of previous
growth medium having nearly similar pH. This could be attributed experiment (reduction by ~20%). The TDS also reduced further by
to the harsh chemical nature of the lime liquor as compared to pure 6% as compared to previous experiment. However when pellets
growth medium, which could have stunted the growth of the were re-suspended in fresh growth medium and then added to
selected isolate. Here, TDS was monitored every alternate day and lime liquor, it showed exceptionally good results with a reduction
was found to reduce by 18%, 37%, 44% and 49% after 2, 4, 6 and 8 in pH from 13.5 to 8.65 (~34%) within the same time period. The
days respectively. The experiment was repeated twice and the TDS also exhibited a further reduction by 10% when measured after
average of this data is represented here. In order to further curtail a period of 8 days (Fig. 1). The newly adopted method of inoculum
the time taken for reduction in pH and TDS, optimization studies preparation proved to be quite effective exhibiting a substantial
were carried out. reduction in pH by nearly 35% and TDS by almost 59%.

3.3. Identification of the promising bacteria
Isolate YK-L, used for the present study was observed to be Gram
positive cocci. Further identification based on morphological and
biochemical characteristics assigned it as Micrococcus sp. according
to Bergey's Manual of Systematic Bacteriology (Table 2) which was
further confirmed by 16S rDNA studies. The 16S rDNA sequence of
this strain exhibited 99.6% homology with Micrococcus sp. ZJHD5-
19. The strain was deposited at MTCC, Chandigarh and assigned
number MTCC 5899 (Khambhaty and Ojha, 2014). Sequence is
publically available in the GenBank database under accession
3.5. Improvisation of various parameters for maximum pH and TDS
reduction
3.5.1. Study on varying dilutions of effluent
In this experiment, it was observed that liquors which were
diluted in the ratio of 75: 25, 50:50 and 25: 75 exhibited a drop in
pH and came down to 12.7, 12.1 and 11.5 due to dilution at the initial
stage. When the new method of inoculum preparation was fol-
lowed, the undiluted lime liquor recorded a reduction in pH by
almost 2.5 units (approximately 36%) within 24 h of inoculation.

Table 1
pH and salt tolerance of selected bacterial isolates.

YK-I YK-J YK-K YK-L

pH
6.0 þþ e e -
8.0 þþ þþ ± þþ
10.0 þþþ þþ þ þþþ
11.5 þþ e e þþþ
13.0 þ e e þþ
Salt concentration (%)
0 þþþ þþ þþ þþþ
2 þþþ þþ ± þþþ
4 þþ þ - þþþ
6 þþ þ - þþþ
8 þ - - þ
10 - - - þ

- No growth, ± Moderate growth, þ Growth, þþ Good growth, þþþ Very good Fig. 1. Reduction in pH and TDS of lime liquor by YK-L using different methods for
growth. inoculum preparation.
564 Y. Khambhaty et al. / Journal of Cleaner Production 161 (2017) 560e566

Similar trend was observed for the liquor diluted at 75:25 ratios,
since there was not much dilution at the initial phase. Nevertheless,
for the other two dilutions, pH drop was quite faster as should be
the case due to higher dilution at the initial phase coming down to
almost neutral within just 16 h of inoculation (Fig. S2). For this
experiment, TDS reduction of only undiluted effluent was recorded
at the end of 192 h, and was noted to be 8874 ppm thereby
recording a reduction by 55.63%. The faster reduction in pH due to
dilution of lime liquor paves the way for diluting this liquor with
effluent from other sources for effective discharge and treatment in
CETP (This experiment and all subsequent experiments were
repeated thrice and the average of the three data was considered).

3.5.2. Effect of higher inoculum size

Based on the results of this experiment, it was noted that a
higher inoculum ratio of 100:25 and 100:50 did not give any sig-
nificant decrease in pH (7.8 and 7.6 respectively) as compared to
that of the optimized method where a ratio of 100:10 was main-
tained. However, for inoculum ratio of 100:100 a drastic reduction
in the pH was observed. This might be attributed due to two rea-
sons; first the effluent got diluted by 100% with inoculum resulting
in the initial pH of 11.7 and, second there was addition of huge
amount of fresh inoculum, resulting in more number of bacterial
cells added. However, this was not a practically feasible method and
hence, the optimized ratio (100:10) was maintained for further
experiments (Fig. 2). Total Dissolved Solid was measured only for
the undiluted lime liquor at the end of 192 h, and was noted to be
around 4000 ppm thereby recording a reduction by 57.16%.
3.5.3. Effect of initial TDS levels over time
From the data of previous experiments, it was noted that the
reduction in TDS was directly related to its levels at the initial point.
Keeping this in view, an experiment was designed with two liquors
having TDS of 12,000 and 36,000 ppm. It was observed that the set
with low TDS, took half the time (~4 days) to reduce the TDS levels
as compared to approximately 8 days for the one with 36,000 ppm.
The TDS reduced to around 85% at the end of 8 days in the set with
lower concentration as compared to 55% for higher concentration
(Fig. 3). Hence, the initial TDS levels were found to have a profound
effect on its reduction.
Fig. 3. Effect of initial TDS levels on its reduction by Micrococcus sp.
nitrogen sources, it was observed that glucose (1%) and peptone
(0.5%), best supported the growth of bacterium leading to quick
neutralization in substantially lesser time as compared to other
carbon and nitrogen sources. However, good results were also
observed when yeast extract and sodium nitrate were added
(Fig. S3). The TDS was not measured here, since this experiment
was merely conducted to observe the behavior of different carbon
and nitrogen sources to achieve rapid neutralization.
Attempts were also made to make use of agricultural residues
like Wheat bran, Sugarcane bagasse and Jackfruit seed powder in
lieu of conventional substrates. These were used in combination
with peptone. Comparable results with the conventional substrates
in reducing the pH were observed. However, the combined effect of
Jackfruit seed and peptone gave the maximum neutralization fol-
lowed by the other three (Fig. 4). Hence, the use of agro residue as
an alternative to conventional substrates cannot be denied. This
gives a dual benefit of utilizing the waste and better neutralization
achieved by the bacteria which could grow luxuriantly in the
presence of these residues.
3.6. Bacterial arbitrated neutralization process and comparison
among conventional and bacterial neutralization

3.5.4. Study on the effect of various types and concentration of

When experiment with heat killed and live bacterial cells along
carbon and nitrogen sources
with control (no bacteria added), was conducted, it was noted that
While studying the effect of various conventional carbon and
for control and heat killed bacterial cells, the pH remained

Fig. 2. Effectiveness of varying dilutions of lime liquor and size of inoculum on Fig. 4. Study on utilization of un-conventional carbon sources on pH and TDS reduc-
reduction in pH and TDS by Micrococcus sp. tion by Micrococcus sp.
 Y. Khambhaty et al. / Journal of Cleaner Production 161 (2017) 560e566
unaltered, where as in the case of live bacterial cells huge reduction
in pH was observed proving that the neutralization was possible
only due to the presence of live bacteria (Fig. S4). When lime liquor
with a TDS of ~20,000 ppm was subjected to conventional
neutralization with acid, the TDS was found to increase by 22.7,
34.18 and 12.31% for hydrochloric, sulfuric and nitric acid respec-
tively. However, for the biologically treated liquor, reduction in TDS
by around 41% was recorded as compared to control (Fig. 5). This
proves that biological neutralization is far better an option of choice
for treating such effluents.
3.7. Analysis of liquor before and after treatment
The BOD5 and COD of lime liquor before and after treatment
were analyzed. A maximum reduction in BOD5 and COD by ~62%
and 65% was observed when Jackfruit seed powder and peptone
were used as additive in the growth medium. Comparable results
were also obtained with the other treatments (Fig. 6).
3.8. Characterization of the metabolic products
In order to have an idea on the metabolic products generated
upon biological neutralization, FT-IR spectroscopy was performed
(Table 3, Fig. S5). From the three data including liquor which was
not added with any carbon source (1), and liquor samples added
with glucose and peptone (2) and jack fruit seed powder and
peptone (3), it was observed that the first two recorded a total of 9
IR transmission peaks each, indicating the complex nature of the
samples. The absorption band at 3750 cm1 was indicative of
asymmetric stretch of water and peaks at 2920/2923, 2855, 2365
and 2316 cm1 were resulted from CeH, symmetric stretch of CeH,
presence of CO2, C^N stretching, in samples (1) and (2). A small
absorbance band of C]O stretch near 1749 cm1, indicated free
fatty acids in (1) and (2). The peaks, observed at 1141 in sample (1)
and 1010 cm1in both (1) and (2), could be attributed to CeO
stretching and OeH deformation bands, respectively (Williams and
Fleming, 1991; Field et al., 2005). The spectrum of (3) recorded
peaks at 3865, 3465, 2937, 1640, 1564, and 1394 cm1, which cor-
responds to eNH stretching of primary amines, water HeOH
stretching, symmetric eCH2e stretching, HeOeH bending vibra-
tion, COO mode of the carboxylate and carboxylate, respectively,
in addition to peak at 3750 cm1. The IR data shows that bacterial
culture supernatant contains compound with carboxylic acid
group.
Fig. 5. Comparative study on fate of TDS by bacterial neutralization with conventional
acid neutralization of lime liquor.
565
Fig. 6. Analysis of lime liquor before and after biological treatment.
Table 3
FT-IR spectroscopy results of the lime liquor treated with Micrococcus sp.
Peak Wave number (cm1) Inference
number
1 3436.74 Hydroxyl
2 2920/2923, 2855, 2365 CeH, symmetric stretch of CeH, CO2,
and 2316 C^N stretching
3 near 1749 free fatty acids
4 1394 carboxylate
5 1141 CeO
6 1010 OeH
4. Discussion
The study on unusual physiology involving tremendous adap-
tation to high pH environment has enabled the development of
diverse applications of alkaliphiles. Majority of alkaliphiles are re-
ported to require Naþ ions for growth. For instance, Bacillus firmus
and Exiguobacterium auranticum use Naþ/Hþ antiporter system in
the region of pH 7.0 to 9.0, with the usual respiration-coupled
extrusion of Naþ ions being replaced by at least 2 antiporter pro-
teins for the uptake of protons (Padan and Schuldiner, 1994). Quite
a few reports exploiting this potential of alkaliphiles are available,
nonetheless this being a first report for lime liquor of tannery. A
process employing two alkaliphilic Bacillus species for the
neutralization of textile industry waste water (pH 12.0 and 10.5)
was described by Kumar and coworkers (Kumar et al., 2005a, 2011;
Kumar and Kumar, 2006). It was observed that, even though carbon
source was added the maximum change in pH by 3.66/2.24 units
was observed only after 7/5 days of incubation respectively. A
similar study utilizing an alkaliphilic Exiguobacterium sp. 12/later
identified as Exiguobacterium alkaliphilum sp. nov. (Kulshreshtha
et al., 2012), was subjected to neutralize alkaline wastewater
from beverage industry. Neutralization was achieved within a short
span of 2 h by production of carboxylic acid (Kulshreshtha et al.,
2010, 2013), which were studied in details (Kulshreshtha et al.,
2012). Another such invention, discussed about the use of bacte-
rial strain of accession No. 5097, which was useful for reducing the
TDS levels from both raw and electro floated tannery effluents,
however, the percentage of reduction was meager even at the end
of 15 days (Kumar et al., 2005). Jain et al. (2011) studied the bio-
logical neutralization of chlor-alkali industrial effluent by an alka-
liphilic Enterococcus faecium, isolated from Gujarat coast. The
isolate could bring down the pH of waste water from 12.0 to 7.0
566 Y. Khambhaty et al. / Journal of Cleaner Production 161 (2017) 560e566
within 3 h in the presence of carbon and nitrogen sources, with
simultaneous reduction in TDS up to 19e22%. The present study
focuses on two aspects viz. neutralization and TDS reduction of
lime liquor using biological means. This method is proved to be far
more eco-friendly and effluent can be safely discharged at CETP's.
With the use of appropriate carbon and nitrogen substrates the pH
as well as TDS levels can simultaneously be brought down in a
substantially lesser time. The change in pH may be controlled by
two factors: a) the production of organic acid or alkaline substances
during the biological metabolism or b) the buffer capacity of the
influent (Lijklema, 1969; Paavilainen et al., 1994). Further, for bio-
logical treatment of such waste waters, prior neutralization is
essential as most of the microbial strains present in activated
sludge process work well near neutral pH. Therefore, alkaline waste
waters need to be first neutralized and then either sent to drainage
or to treatment processes.
5. Conclusions
The present study attempts to address two major issues of the
tannery lime liquor using biological means. Micrococcus sp. was
exploited for the same and reduction in pH and TDS could be
achieved in a reasonably less time. The utilization of agro residues
as cheap carbon source, for the same has also been successfully
explored which opens the possibility of using these waste mate-
rials. This work paves way for a greener, eco-friendly route for the
treatment of highly alkaline waste waters.
Conflict of interest
The authors declare that they have no competing interests.
Acknowledgement
The authors acknowledge the Science and Engineering Research
Board, DST, New Delhi, India for financial grant (SR/FT/LS-166/
2012). CSIR-CLRI Communication No. 1219.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jclepro.2017.05.167.
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