Bioresource Technology 102 (2011) 677–682

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

Removal of sulphate, COD and Cr(VI) in simulated and real wastewater by

sulphate reducing bacteria enrichment in small bioreactor and FTIR study
Rajesh Singh a, Anil Kumar a, Anita Kirrolia a, Rajender Kumar b, Neeru Yadav a, Narsi R. Bishnoi a,⇑,
Rajesh K. Lohchab a
a
Department of Environmental Science & Engineering, Guru Jambheshwar University of Science & Technology, Hisar-125001, India
b
Department of Chemical Science & Engineering, Royal Institute of Technology, SE 100 44, Stockholm, Sweden

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

Article history: The present study was conducted to investigate the chromium(VI), COD and sulphate removal efficiency
Received 14 May 2010 from aqueous solution and treatment of real effluent (CETP) in a small scale bioreactor using sulphate
Received in revised form 16 August 2010 reducing bacteria consortium. Effect of different hydraulic retention times (HRTs), initial metal concen-
Accepted 16 August 2010
trations, various carbon sources and temperatures were studied on removal of chromium(VI), COD and
Available online 24 August 2010
sulphate. Maximum chromium(VI) and sulphate removal was found to be 96.0% and 82.0%, respectively,
at initial concentration of 50 mg l1 using lactate as carbon source. However, highest COD removal was
Keywords:
36.2% in medium containing fructose as the carbon source and electron donor. NADH dependent chro-
Sulphate reducing bacteria
Consortium
mate reductase activity was not observed which indicated the anaerobic consortium. Initially consortium
Chromium medium with a strong negative oxidation reduction potential indicated the reducing activity. The FTIR
Bioreactor spectrum of the sulphate reducing bacteria consortium clearly shows the existence of the sulphate ions
Metal removal and signifies that sulfate reducing bacteria have used sulfate during the growth phase.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction mouth and gut of many animals, including man (Martins et al.,
2009; Matias Pedro et al., 2005; Chang et al., 2001). The ability to
The sources of metal wastes are diverse in nature and geograph- produce sulphide and bicarbonate by sulphate reducing bacteria
ical distribution. The presence of heavy metals in final industrial (SRB) in the presence of a suitable electron donor and carbon
effluents is extremely undesired, as they may accumulate to toxic source helps in the treatment of wastewater. The bicarbonate alka-
levels and cause ecological damage under certain environmental linity neutralizes acidity while dissolved metals are precipitated by
conditions (Pagnanelli et al., 2010). When discharged untreated, the sulphide as summarized in Eqs. (1–3) (Drury, 1999):
it poses a threat to the quality of freshwater resources and conse-
quently the well-being of humans and the environment at large
Electron donor þ SO2  
4 ! HS þ HCO3 ð1Þ
(Oyekola et al., 2010). Anaerobic techniques are becoming increas-
ingly important and intensively studied as they are cost-effective Me2þ þ HS ! MeS # þHþ ð2Þ
and environmentally safe. Biological treatment with sulphate
reducing bacteria (SRB) has been considered as the most promising HCO3 þ Hþ ! H2 O þ CO2 ð3Þ
alternative for the treatment of several types of industrial waste- Due to high mobility and toxicity hexavalent chromium has
waters. Sulphate reducing organisms (SRO) are important mem- gained more importance than trivalent chromium. The toxicity of
bers of microbial communities with economic, environmental chromium is highly dependent on its oxidation state. Hexavalent
and biotechnological interest as they can exist in a variety of envi- chromium is known to be toxic to many plants, animals and micro-
ronments such as soil, sediments and domestic, industrial and organisms inhabiting aquatic environments, but if reduced to chro-
mining wastewaters, marine and fresh waters as well as in the mium(III) it may be significantly less harmful. The studies
performed with chromium(VI) bioaccumulating microorganisms
(Donmez and Aksu, 2002; Dursun et al., 2003; Baldrian, 2003;
Abbreviations: ORP, oxidation reduction potential; COD, chemical oxygen
Zouboulis et al., 2004; Donmez and Koc-berber, 2005) have shown
demand; SRB, sulphate reducing bacteria; NADH, nicotinamide adenine dinucleo-
tide; Cr(VI), hexavalent chromium; FTIR, Fourier transforms infrared spectroscopy. that microbial chromium(VI) removal from solutions typically in-
⇑ Corresponding author. Tel.: +91 01662 263321. cluded the following stages: (1) the binding of chromium to
E-mail address: [email protected] (N.R. Bishnoi). cell surface, (2) translocation of chromium into the cell, and (3)

0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.08.041
678 R. Singh et al. / Bioresource Technology 102 (2011) 677–682

reduction of chromium(VI) to chromium(III) two reaction steps were performed in duplicate using Postgate growth medium hav-
have been suggested to involved in the reduction reactions (Suzuki ing pH around 7. The bioreactors were connected with 500 ml aspi-
et al., 1992). First Cr(VI) accepts one molecule of NADH and gener- rated bottles (gas holder) filled with water to trap the gases
ates Cr(V) as an intermediate (Eq. (4)), and then Cr(V) accept two generated during growth. The gas holder outlet was discharged
molecule of electron to form Cr(III) (Eq. (5)). into another aspirated bottle to handle the displaced volume of
the water in the gas holder. All the reactors were made airtight,
Crþ6 þ e ! Crþ5 ð4Þ which were checked by the drop in the water level in the gas
holder. The reactors containing 400 ml Postgate growth media
Crþ5 þ 2e ! Crþ3 ð5Þ amended with 50 mg l1 of Cr(VI) was inoculated with 100 ml of
The overall Bio-reduction of Cr(VI) and precipitation of Cr(III) inoculums and incubated at various temperature ranging from 30
might be illustrated by following Eqs. (6) and (7) and under anaer- to 40 °C in the BOD incubator for 3 days under the static condition.
obic conditions when glucose as electron donor, the Cr(VI) micro- The effect of various carbon sources was studied using the Postgate
bial reduction might be expressed as (Eq. (8)) (Chen and Gu, 2005). growth medium amended with different carbon sources, like so-
dium lactate, glucose, sucrose and fructose. The different carbon
CrO2 þ 
4 þ 8H þ 3e ! Cr
þ3
þ 4H2 O ð6Þ sources were amended in such a way that total carbon content
was approx 2.58% from the various carbon sources. The glucose,
Crþ3 þ 4H2 O ! CrðOHÞ3 þ 3Hþ þ H2 O ð7Þ fructose, sucrose and lactate added were 5.0 gm/l, 5.0 gm/l,
5.9 gm/l and 15 ml/l, respectively.
C6 H12 O6 þ 8CrO2   For wastewater treatment 100 ml sulphate reducing bacteria
4 þ 14H2 O ! 8CrðOHÞ3 þ 10OH þ 6HCO ð8Þ
innoculum was inoculated in the 400 ml mixture in 1:1 ratio (v/
Although it is well established that SRB can reduce different v) of Postgate growth media and wastewater in the reactors. This
metals, this paper presents a study to determine the major factors mixture was inoculated with 100 ml of the SRB innoculum and
affecting biological treatment of Cr(VI) contaminated simulated amended with 50 mg l1 Cr(VI). The wastewater treatment study
wastewater. was carried out in duplicate and the mean value was reported.
The main objective of this research work was to operate the sul- The reactors were kept in BOD incubator at optimized conditions
phate reducing consortium in a controlled bioreactor system, to for 3 days under static conditions. Sampling from the reactors
determine the sulfate reduction rate, COD removal, chromium re- was performed at 0 h and 3 days of incubation.
moval and pH profile. The small scale reactor was selected, as we
can control all the influential environmental parameters in well or- 2.3. Chromate reductase enzyme assay
ganized manner. The optimized conditions were applied for real
wastewater treatment and FTIR study. The optimum conditions The samples withdrawn from the reactors were also used for
evaluated under small scale reactor can further be applied at pilot Cr(VI) reductase enzyme assay as described by Elangovan et al.
scale bioreactors. (2006). The samples for the enzyme assay were centrifuged at
7500 rpm at 4 °C for 10 min in 1.5 ml centrifuge tube. Chromate
2. Methods reductase activity was determined by measuring the decrease in
hexavalent chromium by the s-diphenylcarbazide method in the
2.1. Isolation and growth media for sulphate reducing bacteria presence of NADH as an electron donor. The assay was carried
out in 1.5 ml reaction mixture containing 100 ll crude enzyme,
The search for sulfate reducing bacteria was done by using the 500 ll Cr(VI) (5 lg/ml), 500 ll NADH (0.1 mM) and 400 ll phos-
anaerobic digester sludge collected from sewage treatment plant. phate buffer (pH 6). The assay mixtures were incubated at 30 °C
The sludge sample was screened for the removal of big particles. for 30 min. The enzyme blank was carried out without NADH to
One gram of the screened sludge was inoculated in small scale bio- find out the residual hexavalent chromium in the medium. The
reactor containing 500 ml Postgate (1984) isolation selective med- reaction mixture was diluted to 10 ml and 0.1 ll concentrated
ium. The composition of the Postgate isolation media was as H2SO4 was added. To find residual hexavalent chromium, 0.2 ml
(g l1): Na2SO4 1.0; KH2PO4 0.5; NH4Cl 1.0; CaCl2 0.7; FeSO4 0.1; of the s-diphenylcarbazide reagent (dissolved in acetone) was
yeast extract 1.0; agar 15.0; mercaptoacetic acid 0.1; ascorbic acid added to final 10 ml reaction mixture and kept for developing color
0.1; ethanol (absolute) 4.5 ml; and Resazurin 0.1%. Reactor was before taking OD540nm.
kept in anaerobic conditions for the growth of anaerobic bacteria
for a period of 7 days at 37 °C till the colour of media changed to 2.4. Analysis for various parameters
blackish grey. After 7 days of anaerobic incubation, the 100 ml
inoculum of isolates was further transferred in 400 ml sterilized Ten millilitre of the samples were withdrawn from the bioreac-
Postgate growth medium in another 500 ml reactor. The composi- tors and centrifuged at 5000 rpm to prepare the cell free superna-
tion of the Postgate growth medium used was as (g l1): KH2PO4 tant at 4 °C. This supernatant was used for further analysis of
0.5; Na2SO4 1.0; NH4Cl 2.0; CaCl2 0.06; FeSO4 0.005; sodium citrate various parameters. The soluble chemical oxygen demand in the
0.3; yeast extract 0.1; sodium lactate 15 ml. The Postgate medium supernatant was measured by the Spectralab COD Digester
is partially selective for SRB, with sodium lactate as electron donor (2015 M) and COD Titrator (CT-15) using the Platinum combined
and carbon source. The indication of successful incubation was the electrode. Total residual chromium was quantified by Atomic
blackening of the medium, and H2S generation recorded as positive Absorption Spectrophotometer (Shimadzu AA-6300, Japan). The
for SRB presence. sulphate in the diluted sample was measured using standard
methods of APHA-1995. Total organic carbon was measured by
2.2. Batch experiments wet oxidation followed by titration with ferrous ammonium sul-
fate using orthophenanthroline ferrous complex (Ferroin) indica-
Static batch anaerobic sorption experiments were conducted in tor. pH of the withdrawn samples was measured immediately
500 ml (total volume) designed circular glass reactors having one without centrifugation using EUTECH Instruments (pH Tutor). Oxi-
mouth provided with screw cap with connection systems, two in- dation reduction potential (ORP) was monitored online using pH
lets for probe insertion and one outlet for sampling. Experiments 1500 Cyberscan (EUTECH Instruments) by inserting the electrode
 R. Singh et al. / Bioresource Technology 102 (2011) 677–682 679

in the one arm of the reactor. The electrode was standardized using 3.2. Carbon substrate utilization by the mixed culture of sulfate
the appropriate buffer before measurement. The oxidation reduc- reducing bacteria
tion potential measurement as millivolt (mV) was carried out at
room temperature. Cultivating the sulfate reducing bacteria consortium as chro-
mium Cr(VI) removal source was shown to be impossible without
supplementing the medium with a carbon source. The modified
2.5. Fourier transforms infrared (FTIR) spectroscopy
Postgate growth medium was tested by varying various carbon
sources such as sodium lactate, glucose, fructose and sucrose each
FTIR spectrum study was carried out to explain the change in
with 2.58% carbon content. Experiments were conducted to com-
the functionalities of the sulphate reducing bacteria consortium
pare the sulphate reduction, soluble COD removal, chromium re-
in the presence of effluent and control (without effluent). The spec-
moval efficiency and pH profile in the presence of the four
tra were collected using PerkinElmer spectrum BX FTIR system
carbon sources (Table 1). The lowest chromium(VI) removal
(Beaconfield Buckinghamshire HP9 1QA) equipped with diffuse
58.4% and 68.2% was obtained with fructose and glucose, respec-
reflectance accessory with the range of 400–4000 cm1. All spectra
tively. The substrate fructose, sucrose and glucose could be fer-
were acquired in transmission mode, by the KBr disc method. To
mented rather than oxidized and support the fermentative
get the information specific to the functional group, and also on
bacteria rather than reducing bacteria. The maximum chro-
the interaction of the groups with other parts of the molecule
mium(VI) 96.7% removal was obtained with lactate as carbon
and on the spatial properties of the group by FTIR, the study for
source. This is perhaps due to the oxidization of lactase by the
the sulphate reducing bacteria consortium was carried out by
SRB consortium. The reduction in pH of the media to acidic condi-
holding temperature 37 °C under static conditions in the reactors.
tions, supported by fructose, sucrose and glucose as carbon sources
The effluent treatment experiments for FTIR study were carried
also indicated that fermentation occurs when the consortium was
out for 3 days under static conditions. The control of the consor-
supplemented with these carbon sources.
tium was also run parallel in the Postgate growth medium. Cells
Utilization of sugars is still an unusual feature among the sulfate
were harvested after 3 days of incubation by centrifugation at
reducing bacteria (Azabou et al., 2005). Based on the kinetic data,
5000g. Effluent loaded consortium cells and control cells pelleted,
fermentative bacteria can successfully compete with SRB for sugars
were washed with double distilled water to remove the loosely
and amino acids, whereas acetogenic bacteria can effectively com-
bonded ions or impurities and again centrifuged to collect the
pete with SRB for intermediate fermentation products, even in ex-
washed cells. The biomass collected was dried at 50 °C in a heating
cess of sulfate (O’Flaherty and Colleran, 1999). From table, we can
air oven. The samples were grounded in an agate pestle and mortar
conclude that when sodium lactate was used as a carbon source
with KBr. The background obtained from KBr disc was automati-
at optimized temperature, maximum reduction in sulphate was
cally subtracted from the sample discs spectra prepared with
about 82.5% followed by glucose 70% and least 12.45% in the su-
KBr. All spectra were plotted using the same scale on the transmit-
crose. Maximum sulphate reduction in the lactate medium was
tance axis.
due to the alkaline condition produced by the SRB consortium. pH
of the medium plays an important role, when sulfate is reduced
to sulfide; the sulfide can be present in different forms like H2S,
3. Results and discussions
HS and S2 (Al-Zuhair et al., 2008; Tang et al., 2009). Under neutral
conditions most of the sulfide concentration is as hydrogen sulfide
The efficiency of the process was assessed through determina-
(Perry and Green, 1984). It has been shown that at pH less than 7.0,
tion of pH, sulphate, metals ions, COD removal in the simulated
undissociated H2S is the dominant inhibitor. Martins et al. (2009),
wastewater and real wastewater effluent. Formation of black pre-
explained that there will be 75% reduction in sulphate, partial re-
cipitate are indicative of sulphide as a reduction of sulphate
moval of Fe, Zn and complete removal of Cu took place when sul-
(SO2
4 ) to sulphide (S
2
) (Herbert and Gilbert, 1984; Hamilton,
phate reducing bacteria used lactate as a carbon source. At low
1994).
pH the hydrogen sulfide produced exists in undissociated form
and with increase in pH it dissociates into HS and S2.
3.1. Determination of optimal chromium concentration for growth of The most efficient sulphate reduction was generally observed
sulfate reducing bacteria with lactate, due to its total consumption. In case of sucrose and
fructose the pH of the medium was 4.41 and 4.46 indicating en-
Thirty millilitre culture tubes containing 25 ml Postgate growth hanced concentration of H+ ions. The sulfide concentration ap-
medium were supplemented with increasing concentrations of proach a saturation concentration at the constant pH 4 attained
chromium(VI) from 25 to 250 mg l1 using lactate as carbon in the solution and abundance of protons present in solution at
source. Culture tubes were seeded with 1 ml inoculum from the these acidic pH values prevented the speciation of the H2S (aq) into
enrichment culture (Master culture) and incubated at 37 °C under HS (Karbanee et al., 2008).
static conditions for 1 week in BOD incubator. The culture tubes
were tightly caped to prevent the aeration. The change in colour 3.3. Effect of incubation temperatures on the activity of SRB
from yellowish to colourless indicated the growth of the consor- consortium
tium. Growth of sulfate reducing bacteria was monitored by mea-
suring O.D620nm. All experiments were performed in duplicate and The operating temperature plays important role in the micro-
results were expressed as the mean value. Initially, increasing dose bial growth, kinetics of organic substrate decomposition, as well
of chromium(VI) up to 50 mg l1 increased the microbial growth
Table 1
indicated that chromium acts as essential nutrient for growth.
Effect of carbon sources on various parameters.
The maximum OD620nm observed was 1.3 at 50 mg l1 Cr(VI) con-
centration. Beyond 50 mg l1 of Cr(VI), the microbial growth was Parameters Glucose Sucrose Fructose Lactate

inhibited due to toxicity and after 150 mg l1 no sufficient growth pH 5.99 4.41 4.67 8.03
was observed by the SRB. Thus 50 mg l1 concentration was con- % COD removal 36.2 18.7 32.8 16.1
% Sulfate removal 70.3 12.45 31.2 82.5
sidered as the optimum metal loading dose for further
% Chromium removal 68.2 71.2 58.4 96.7
experiments.
680 R. Singh et al. / Bioresource Technology 102 (2011) 677–682

as on hydrogen sulphide solubility of sulphate reducing bacteria 3.4. Effect of hydraulic retention time
(Neculita et al., 2007). To find out the optimum temperature for
the isolated consortium, experiments were performed at different Effects of hydraulic retention time on efficiency of bioreactors
temperatures viz 30, 35, 37 and 40 °C for 3 days incubation period. have been widely studied. It was usually accepted that precipita-
The optimum growth temperature for mixed culture of sulfate tion of metal sulfides occurred in at least 3–5 days (Kuyucak and
reducing bacteria grown with lactate as sole source of carbon St-Germain, 2006). A shorter HRT may not allow adequate time
was 37 °C. There was 12.0%, 16.6%, 30.9% and 11.6% reduction in for SRB activity to neutralize acidity and precipitate metals. A long-
COD at 30, 35, 37 and 40 °C, respectively (Table 2). It can be con- er HRT may imply depletion of either the available organic matter
cluded from the results that at 37 °C, there will be maximum source or the sulfate source for SRB (Dvorak et al., 1992). The reac-
growth of SRB and maximum reduction in parameters. As unex- tors were setup for 7 days at optimum conditions 37 °C with so-
pected, the fastest drop in sulfate concentration was observed at dium lactate as carbon source. From the Fig. 1 we can conclude
40 °C, the drop in sulfate concentration at 40 °C was higher than that pH of the reactor fluctuate in between 6.5 and 8.1, which
that at 30 °C. This can be due to increase in the maintenance coef- was optimum for the activity of the SRB. Generally, SRB are less
ficient with temperature, where all the substrate are utilized for sensitive to total sulfide when the pH is increased from 6.8 to 8.0
the survival of bacteria, and less amount are utilized for the growth and more sensitive to the undissociated sulfide concentration. As
(Al-Zuhair et al., 2008). the pH increases, lesser concentration of undissociated H2S is
The variation in the pH profile of growing consortium may be needed to inhibit the growth. The continuous COD reduction was
the reason for differential removal of sulphate at various tempera- observed and 81.7% was achieved after 132 h of incubation period.
tures (Table 2). The concentration of undissociated H2S required to 76.8% chromium(VI) removal was observed after 72 h and 84.5%
cause 50% inhibition at pH 6.8–7.2 was relatively constant and, sulphate removal was observed in 144 h of incubation period.
above pH 7.2, the H2S concentration causing inhibition decreased
and only very low levels were required to cause 50% inhibition at 3.5. Monitoring of oxidation reduction potential (ORP) reactions
pH 8.0 and above. However, at higher pH levels, SRB were consid-
erably less sensitive to sulphide inhibition (O’Flaherty et al., 1998). The reactivities and mobilities of various elements including
The levels of undissociated H2S required for 50% inhibition of the heavy metals in biological system depend upon the redox reaction
bacterial groups were found to be lower than total sulphide IC50 conditions. Monitoring oxidation reduction reaction as a result of
value, indicating that H2S was clearly the most toxic form of sul- potential difference in the solution is the best application of ORP
phide (Speece, 1983). measurements (Fig. 2). The ORP measurement was carried out at
room temperature (approx 32 °C), under static conditions. One
week fully grown consortium was amended with 50 mg l1 chro-
mium(VI). When an oxidation–reduction reaction goes to comple-
Table 2 tion, there is usually a large change in the ORP. The typical purpose
Effect of incubation temperature on reduction of various parameters. of using ORP is to ensure that an ORP reaction has gone to comple-
tion, i.e., the substance of interest has been completely reduced or
Parameter Temperatures
completely oxidized. ORP increased only slightly during the initial
30 °C 35 °C 37 °C 40 °C
period of incubation as inferred from series 1, 2, 3 and 4 (Fig. 2).
pH 8.13 6.71 7.16 6.92 During this period change in the ORP is very small and hence very
% COD removal 12.0 20.4 34.09 23.74
poor reduction of Cr(VI) takes place. The ORP change after 140 h of
% Sulfate removal 20.5 54.9 29.3 92.7
% Cr (VI) removal 78.2 83.1 96.8 56.6
incubation is quite large, beyond the point, the ORP continuously
dropped to 153.6 mV. This point represents the equivalent point

pH % COD Removal % Sulphate removal % Cr(VI) Removal

pH
33
38
47
08
83
53
77
81
16
77

53
03
06

42
57
39

83
31
3

1
7.
7.
7.
7.
7.
6.
6.
6.
6.
7.
6.
7.

8.
8.
7.
7.
7.
7.
8.
7.
7.

90 90
80 80
70 70
% Removal

60 60
% Removal

50 50

40 40
30 30
20 20
10 10
0 0
12
18
24
30
36
42
48
54
60
66
72
78
84

8
0
2
4
8
0
6

96
10

14
12
13

16

Hydraulic retention time (hrs)

Fig. 1. Effect of hydraulic retention time on various parameters pH (secondary X axis), COD removal, sulphate removal and Cr (VI) removal by SRB consortium. The values
presented on the secondary X axis indicated the pH values observed.
 R. Singh et al. / Bioresource Technology 102 (2011) 677–682 681

1 2 3 4 5 6 7 The increase in pH of the effluent from 6.97 to 7.57 indicated

the growth of sulfate reducing bacteria due to formation of the hy-
400
droxyl ions. At this pH, SRB were capable of removing 90% sulfate
from the wastewater in an anaerobic bioreactor operated under
350 alkaline condition with lactate as organic carbon source. Sulfate re-
moval was the best indicator of SRB activity (Johnson and Hallberg,
2005). The sulfate reduction reaction was capable of contributing
300
HCO 3 alkalinity to the system to maintain an optimal pH range.
During bio-treatment, acidity removal and effluent alkalinity were
250 due to bicarbonate produced by bacterial sulfate reduction (Dvorak
- ORP (mV)

et al., 1992). The alkalinity before treatment in the effluent was

900 mg l1 while after treatment it increased to 2950 mg l1 due
200
to the formation of bicarbonate because of SRB activity. Anaerobic
treatment of Okhla industrial area effluent (CEPT) by strains iso-
150 lated from the sludge improved the COD removal to 13.5%. The re-
moval of organic components was mainly accomplished by sulfate
reducing bacteria which utilized organic substrate from the waste-
100
water as a carbon source. In addition, sulfate reduction can also
help in eliminating some fraction of organic substances. By
50 increasing the pH of acidic water metals can be removed efficiently
due to precipitation in the form of hydroxides. The metals ions re-
moval efficiency order was Cd2+ > Zn2+ > Pb2+ > Ni2+. Cadmium,
0
zinc, lead and nickel were removed 86.6%, 54.09%, 49.8% and
Time (every intervel 30 minutes) 10.3%, respectively. The higher removal of Cd2+ was due to its
Fig. 2. Behavior of the oxidation redox potential accompanying an oxidation–
strong binding tendency and lesser amount in the effluent. The re-
reduction (series 1, 2, 3, . . . ,7 represents initial 15 h, next 15 h and so on sults indicated that removal efficiency was higher for cadmium
respectively). then nickel by sulphate reducing bacteria. Oil and grease present
in the wastewater was removed up to 75% by SRB.
of consortium. The small decrease in ORP after sharp drop may be
due to shaking of the reactor. Depressions in the other series ob-
3.7. Characterization of functionalities of sulphate reducing bacteria
served may also be due to manually shaking of the reactor. How-
consortium using Fourier transforms infrared (FTIR) spectroscopy
ever, there were no depressions in initially 60 h of incubation
period, this may be due to duration required by the SRB consor-
The FTIR difference spectra in pure SRB consortium was com-
tium for environmental acclimatization in the reactor.
pared to the spectra obtained in effluent loaded biomass to deter-
During the oxidation reduction potential measurement the
mine differences due to interaction of the metals ions and
chromate reductase activity was also studied. The reactor was
contaminants with functional groups. The major stretching were ap-
manually shaken during sampling for chromate reductase activity
peared at 1020.78, 1077.96, 1150.28, 1234.47, 1394.63, 1454.61,
measurement. The cell free supernatant was used to measure the
1539.74, 1652.01, 2367.83, 2928.22, 2958.79 and 3310.44 cm1
chromate reductase activity. The NADH dependent chromate
for control consortium and 1029.75, 1079.11, 1231.63, 1400.37,
reductase activity was absent during growth in the consortium
1457.62, 1542.80, 1654.47, 2373.86, 2856.28, 2975.95 and
supported the anaerobic conditions in the culture medium. As
3310.38 cm1 for effluent loaded consortium. The comparative re-
maximum NADH dependent chromate reductase activity exists
sults showed that the correlation between the two spectra was
with soluble protein produced under aerobic condition.
0.9277 by the factor 0.2506. The effect of effluent loading on the sul-
phate reducing microbial consortium can be clearly inferred the FTIR
3.6. Study of SRB on real wastewater
spectrum. The spectrums were characterized by appearance of the
much sharper peaks in the wave regions 875–1650 cm1. Presence
Physicochemical analysis results indicated that Cr(VI) was be-
of neutral C@O complex is indicated by their stretching modes at
low the detection limit and hence the effluent was amended with
1652.01 cm1 in pure biomass consortium and a slight shift to
50 mg l1Cr(VI) final concentration (Table 3).
1654.47 cm1. Bands are mainly due to phosphate stretching vibra-
Table 3 tion in polysaccharides and nucleic acids in the range 1250–
Effect of treatments on the wastewater partially simulated with Cr 1200 cm1 and 1200–900 cm1 spectral region corresponding to
(VI) dosing. C–O–C and C–O–P. These stretching vibrations imply oligo- and
Parameters Before treatment After treatment polysaccharides present in the bacteria (Rubio et al., 2006). Previous
pH 6.97 7.57
spectroscopic studies report that sulfate ions present five bands cen-
COD 3904 3376 tered at 1230 cm1, 1130 cm1, 1070 cm1, 1000 cm1, and
TOC 270.4 221.0 610 cm1 (Nakamoto, 1970). The spectra of the SRB consortium
Sulphate 2100 212.0 (without effluent) clearly indicated the existence of the sulphate
Alkalinity 900 2950
ions at the similar bands. This signifies that sulfate reducing bacteria
Oil and grease 2.4 0.6
Chromium 50** 82.6* have used sulfate during the growth phase.
Lead 0.702 49.8*
Nickel 0.29 10.3*
Zinc 0.61 54.09* 4. Conclusion
Cadmium 0.03 86.6*

All the parameters are (mg l1) except pH.

The study concludes that maximum removal of chromium was
*
Indicated % removal. found to be 96.7% and 82.6% in simulated wastewater and chro-
**
Amended Cr(VI). mium amended wastewater, respectively. Initial 50 mg l1 of
682 R. Singh et al. / Bioresource Technology 102 (2011) 677–682

Cr(VI) loading with sodium lactate as carbon source for 3 days of Z., Flemming, H.C. (Eds.), Biofouling and Biocorrosion in Industrial Water Systems,
second ed. CRC Press Inc., Boca Raton (FL), pp. 27–36.
HRT, signified that consortium was less affected due to the toxicity
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