Journal of Hazardous Materials B126 (2005) 78–85

Biological chromium(VI) reduction using a trickling filter

E. Dermou a , A. Velissariou b , D. Xenos a , D.V. Vayenas a,∗
a Department of Environmental and Natural Resources Management, University of Ioannina, Seferi 2, 30100 Agrinio, Greece
b Hellenic Aerospace Industry S.A., P.O. Box 23, GR 32009, Schimatari, Greece

Received 27 January 2005; received in revised form 2 June 2005; accepted 2 June 2005
Available online 27 July 2005

Abstract

A pilot-scale trickling filter was constructed and tested for biological chromium(VI) removal from industrial wastewater. Indigenous bacteria
from industrial sludge were enriched and used as inoculum for the filter. Sodium acetate was used as carbon source and it was found to inhibit
chromate reduction at high concentrations. Three different operating modes were used to investigate the optimal performance and efficiency
of the filter, i.e. batch, continuous and SBR with recirculation. The latter one was found to achieve removal rates up to 530 g Cr(VI)/m2 d,
while aeration was taking place naturally without the use of any external mechanical means. The low operating cost combined with the high
hexavalent chromium reduction rates indicates that this technology may offer a feasible solution to a very serious environmental problem.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Chromate; Biological removal; Trickling filter; Sequencing batch reactor; Recirculation

1. Introduction low-pH conditions and subsequent adjustment of solution

pH to near neutral ranges to precipitate Cr(III) as hydroxides
Chromium is one of the most toxic heavy metals dis- [5]. However, this method is not completely satisfactory
charged into the environment through various industrial because of the large amount of secondary waste products due
wastewaters, and has become a serious health problem. Metal to various reagents used in the above-mentioned processes.
plating, tanneries and industrial processes using catalysts Biological treatments arouse great interest because of their
discharge worldwide huge amounts of chromium every lower impact on the environment as opposed to chemical
year. The effluents of these industries contain Cr(VI) and treatments. Recent studies have shown that certain species of
Cr(III) at concentrations ranging from tenths to hundreds bacteria are capable of transforming hexavalent chromium,
of milligrams/liter. While Cr(VI) is highly toxic and is Cr(VI), into the much less toxic and less mobile trivalent
known to be carcinogenic and mutagenic to living organisms form, Cr(III) [6,7]. Bacteria may protect themselves from
[1], Cr(III) is generally only toxic to plants at very high toxic substances in the environment by transforming toxic
concentrations and is less toxic or non-toxic to animals [2]. compounds through oxidation, reduction or methylation into
The discharge of Cr(VI) to surface water is regulated to more volatile, less toxic or readily precipitating forms.
below 0.05 mg/l by the US EPA [3] and the European Union The processes by which microorganisms interact with
[4], while total Cr, including Cr(III), Cr(VI) and its other toxic metals enabling their removal/and recovery are
forms, is regulated to below 2 mg/l [3]. bioaccumulation, biosorption and enzymatic reduction
At present, the most commonly used technology for [8]. Microbial heavy metal accumulation often comprises
treatment of heavy metals in wastewaters is chemical of two phases. An initial rapid phase involving physical
precipitation. Conventional chemical treatment involves adsorption or ion exchange at cell surface and by a subse-
reduction of Cr(VI) to Cr(III) by a reducing agent under quent slower phase involving active metabolism-dependent
transport of metal into bacterial cells [9]. Biosorption is a
∗ Corresponding author. Tel.: +30 26410 39517; fax: +30 26410 39576. metabolism-independent process and thus can be performed
E-mail address: [email protected] (D.V. Vayenas). by both living and dead microorganisms. This adsorption is

0304-3894/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jhazmat.2005.06.008
 E. Dermou et al. / Journal of Hazardous Materials B126 (2005) 78–85 79

based on mechanisms such as complexation, ion exchange, 7H2 O, 0.001 g CaCl2 ·2H2 O, 5 g CH3 COONa·3H2 O and
coordination, adsorption, chelation and microprecipitation, 0.5 g K2 HPO4 in 1.0 l of tap water.
which may be synergistically or independently involved [10].
Enzymatic reduction of Cr(VI) into Cr(III) is believed to be 2.2. Reagents
one of the defense mechanisms employed by microorganisms
living in Cr(VI)-contaminated environments. The reduced Stock Cr(VI) solution (500 mg/l) was prepared by dissolv-
Cr(III) may precipitate as chromium hydroxide in neutral pH ing 141.4 mg of 99.5% K2 Cr2 O7 , previously dried at 103 ◦ C
range [11]. for 2 h, in Milli-Q water and diluting to 100 ml. Diphenyl
Most of the previous studies on biological reduction of carbazide solution was prepared by dissolving 250 mg of
Cr(VI) were conducted in batch reactors (flasks) using mainly 1,5-diphenylcarbazide in 50 ml of HPLC-grade acetone and
pure cultures. For instance, Wang and Xiao [12] studied storing in a brown bottle. Potassium hydrogen phthalate stan-
several factors affecting hexavalent chromium reduction in dard (KHP) was prepared by dissolving 425 mg in distilled
pure cultures of bacteria in flasks. Wang and Shen [5] studied water and diluting to 1000 ml. Digestion solution was pre-
the kinetics of Cr(VI) reduction by pure bacterial cultures pared by dissolving 10.216 g K2 Cr2 O7 , previously dried at
in flasks. Shakoori et al. [13] isolated a dichromate-resistant 103 ◦ C for 2 h, in 500 ml distilled water, 167 ml conc. H2 SO4
Gram-positive bacterium from effluent of tanneries and used and 33.3 g HgSO4 and diluting to 1000 ml (for the determi-
flasks as batch reactors. Fein et al. [14] used pure bacterial nation of the COD values).
cultures in flasks to study the non-metabolic reduction of 1,5-Diphenylcarbazide was purchased from Fluka Chem-
Cr(VI) by bacteria under nutrient-absent conditions. Srinath ical, potassium dichromate was purchased from Sigma
et al. [8] studied Cr(VI) biosorption and bioaccumulation Chemical Co. All the others chemicals were purchased from
by pure cultures of chromate resistant bacteria in flasks. Riedel-de Haen.
Megharaj et al. [15] studied hexavalent chromium reduction
in flasks, by pure cultures of bacteria isolated from soil 2.3. Analytical methods
contaminated with tannery waste.
Recently, continuous-flow and fixed-film bioreactors During all experiments, hexavalent chromium concen-
were also used for biological reduction of Cr(VI). Shen and tration, pH, temperature, dissolved oxygen concentration
Wang [16] demonstrated Cr(VI) reduction in a two-stage, and TOC measurements were made on a daily basis.
continuous-flow suspended growth bioreactor system. Samples were filtered through 0.45 ␮m –Millipore filters
Escherichia coli cells grown in the first-stage completely (GN-6 Metricel Grid 47 mm, Pall Corporation). Hexavalent
mixed reactor were pumped into the second-stage plug-flow chromium concentration was determined by the 3500-Cr D
reactor to reduce Cr(VI). Chirwa and Wang [11] demon- Colorimetric method according to Standard Methods for the
strated the potential of fixed-film bioreactors for Cr(VI) Examination of Water and Wastewater [17]. Total organic
reduction. This was the first report on Cr(VI) reduction carbon measurements (TOC) were conducted in order to
through biological mechanisms in a continuous-flow determine the feed sodium acetate concentration both in
laboratory-scale biofilm reactor without the need to con- the liquid culture (chemostat) and the liquid volume of the
stantly resupply fresh Cr(VI)-reducing cells. Bacillus sp. was bioreactor, following the methods described in Standard
used in this work for the transformation of Cr(VI) into Cr(III). Methods for the Examination of Water and Wastewater
Virtually all the previous studies on biological reduction [17] by using, Total organic carbon analyzer (TOC-VCSH ,
of Cr(VI) were conducted in laboratory scale apparatus SHIMAZDU Corporation, Japan). Total chromium con-
(reactors), using sterilized conditions and pure cultures of centration measurements were made according to Standard
microorganisms. The present study is the first to report on Methods for the Examination of Water and Wastewater [17]
Cr(VI) biological reduction in a pilot-scale trickling filter using an atomic absorption spectrophotometer (model AAS-
using mixed culture of microorganisms, originating from 700, Perkin-elmer) (results not shown for total chromium
an industrial sludge. The operation of the trickling filter as concentrations).
a Sequencing Batch Reactor (SBR) with recirculation led
to significantly high Cr(VI) reduction rates, thus promising 2.4. Isolation and enrichment of indigenous bacteria
a feasible technological solution to a serious environmental
problem. Samples of industrial sludge were taken from the Hellenic
Aerospace Industry S.A. In order to grow bacterial strains
able to reduce hexavalent chromium, a sludge sample of 10 g
2. Materials and methods was added in a 2 l Erlenmeyer flask and was diluted in an
acetate-minimal medium and concentrated chromium solu-
2.1. Media tion (in the form of K2 Cr2 O7 ) resulting in a final hexavalent
chromium concentration of 50 mg/l. The final volume of the
The influent feed to the bioreactor was prepared by solution was 1 l. Acetate-minimal medium (AMM) was com-
dissolving 1 g NH4 Cl, 0.2 g MgSO4 ·7H2 O, 0.001 g FeSO4 · prising (per litre) 1 g NH4 Cl, 0.2 g MgSO4 ·7H2 O, 0.001 g
80 E. Dermou et al. / Journal of Hazardous Materials B126 (2005) 78–85

FeSO4 ·7H2 O, 0.001 g CaCl2 ·2H2 O, 5 g CH3 COONa·3H2 O, At the top of the filter, there was a fixed nozzle, which
0.5 g yeast extract and 0.5 g K2 HPO4 in 1.0 l of tap water distributed the incoming solution evenly to the whole filter
(micronutrients were supplied using tap water as diluent), surface. The filter was also equipped with an underdrain
while the final pH of the nutrient solution was adjusted to system to collect the treated water and any biological solids
7. The solution was kept under oxic conditions through aer- that would detach from the media in the case of continuous
ation and mixing while nutrients and hexavalent chromium operation mode. Along the filter depth there were 10
were added according to the observed chromium reduction. sampling ports for chromium concentration measurements
An intense chromium reduction was observed after 30 days in the bulk liquid. Thus, it was possible to have an exper-
of cultivation. Bacterial samples taken at this point showed, imental assessment of the chromium concentration along
using 16S rRNA sequencing [18–20], that the dominant bac- the filter depth and control the homogeneity of the bulk
terial species of the mixed culture was Acinetobacter sp. liquid concentration. Filter backwashing was necessary from
Francisco et al. [21] also report that they identified bacte- time to time due to pore clogging from Cr(III) precipitates
ria belonging to the genus Acinetobacter in a chromium- and was performed using high water and air velocities
contaminated activated sludge. The cell density of the liquid upwards.
culture was determined as optical density at 600 nm on a Throughout all experiments water temperature was fairly
JASCO V-530, spectrophotometer. The liquid culture was constant at about 28 ± 1 ◦ C using a heat exchanger, while
used to inoculate the bioreactor at the beginning of its oper- ambient temperature was about 26 ◦ C (room temperature).
ation. The pH ranged from7.2 to 8.87 and the concentration of
the dissolved oxygen in the liquid phase was physically
2.5. Reactor system maintained at a near constant level of 4.5 mg of DO/l in the
reactor. The above measurements were performed using the
The pilot-scale trickling filter (Fig. 1) consisted of a Plex- Hanna pH211 pH meter, and the Hanna HI9143 dissolved
iglas tube, 160 cm high and 9 cm i.d. This pilot-filter height oxygen meter, respectively. The use of a trickling filter
is typical of a full-scale industrial filter. Since it is the load- has the advantage of not requiring an external air supply,
ings (hydraulic and chromium) per unit cross-sectional area since air is naturally convected through the filter due to the
that matter, no scale-up is necessary. The support material temperature difference between the interior and exterior of
was gravel with a mean diameter of 5.5 mm, and specific the filter. Mechanic aeration was added at the bottom of the
surface area of 1059 m2 /m3 , while the depth of the support filter only under batch operation.
media was 143 cm and the filter porosity 0.4. The gravel was The pilot-scale trickling filter was first operated as a
brought from a beach of Lefkas island, Greece. The filter batch reactor, subsequently as a sequencing batch reactor
media was loaded on the filter and washed with water from with recirculation, and finally as a continuous flow reactor.
the supply network for 48 h. The support material was not The recirculation was provided in order to obtain completely
flooded for flow rates up to 3000 ml/min or hydraulic load- mixed flow pattern in the bioreactor, since the formation of
ings up to 680 m3 /m2 d. chromate precipitates was leading to spatial heterogeneity

Fig. 1. Schematic drawing of the pilot-scale trickling filter arrangement.

 E. Dermou et al. / Journal of Hazardous Materials B126 (2005) 78–85 81

and consequently to insufficient exploitation of the filter. The In the first cycle the filter was loaded with 8 mg/l hex-
discrete values of 960, 2400 and 3200 ml/min were tested avalent chromium (in the form of K2 Cr2 O7 ) mixed with the
for the recirculation stream. Since no substantial difference influent feed to ensure bacterial growth. Sodium acetate was
was observed the value of 2400 ml/min was adopted as it used in excess (5 g/l) in order to avoid growth limitation by
was providing efficient filter wetting without flooding. carbon throughout the experiment. After 8 days a 47% reduc-
tion was observed under fully aerobic conditions (Fig. 3). The
hexavalent chromium reduction rate was the highest observed
3. Experimental results up to this point. However, after this point there is a sig-
nificant drop in reduction rate, as observed in preliminary
Before starting biological chromium reduction, an exper- experiments, which leads to a long time required for total
iment was performed in order to investigate possible physic- reduction. Thus, in order to accelerate startup and keep a
ochemical chromium reduction under the specific experi- high reduction rate the system was reloaded at this point.
mental conditions. For this reason the clean filter (without Nutrients and hexavalent chromium were added (as well as
any bacteria) was loaded with nutrients and chromium at a at the beginning of each cycle) to reach the initial concen-
final concentration of 30 Cr(VI)mg/l, under batch operat- tration of 8 mg/l. After another 8 days operation, maximum
ing mode. Mechanical aeration was added to the system and reduction rate and a 69% reduction of hexavalent chromium
for a period of 8-h operation (Fig. 2) no substantial change were observed, while during the third operating cycle the
of the concentration of hexavalent chromium was observed, reduction of hexavalent chromium reached to 82%. During
indicating the absence of physicochemical reduction of hex- the fourth operating cycle the filter reached fully hexavalent
avalent chromium. The addition of sodium acetate did not chromium reduction in only 6 days and at this point we con-
alter the above observation. Therefore, addition of bacte- sidered the startup period completed and the filter ready for
ria was apparently necessary in order to achieve hexavalent full operation.
chromium reduction. Nutrients were added again as well as hexavalent
chromium this time to a concentration of 30 mg/l. This feed
3.1. Batch operation—reactor startup concentration value was used in all experiments as it was con-
sidered an extreme upper limit for the wastewater effluent of
The pilot-scale trickling filter was inoculated with the Hellenic Aerospace Industry. A new series of operating
enriched bacteria from the industrial sludge of the Hellenic cycles was performed, as in Fig. 3, until a minimum period
Aerospace Industry. It was operated as a batch reactor for a of about 19 h was needed for complete reduction (100%
period of about 50 days, to ensure attachment of the bacterial removal) of 30 mg/l hexavalent chromium (Fig. 4). For the
culture to the support media and development of a of biofilm filter diameter of 9 cm or cross-sectional area of 63.62 cm2 ,
layer (startup), and to investigate the maximum chromate filter removal rate for the batch operating mode was about
reduction rate under this operating mode. 18 g Cr(VI)/m2 d.

Fig. 2. Evolution of hexavalent chromium concentration in the clean (with-

out bacteria) pilot-scale trickling filter with the addition initially of air and Fig. 3. Startup of the pilot-scale trickling filter for the biological reduction
in sequence of air and sodium acetate. of hexavalent chromium.
82 E. Dermou et al. / Journal of Hazardous Materials B126 (2005) 78–85

Fig. 4. Operating cycles of the filter under batch operation. Duration of Fig. 5. Operating cycles of the filter under SBR operation with recirculation.
minimum stable cycles: 19 h. Duration of minimum stable cycles: 40 min.

For the of 30 min operating cycle a hexavalent chromium

3.2. SBR operation with recirculation reduction rate of 535 g Cr(VI)/m2 d was achieved. This type
of operating mode accomplished the highest reduction rate,
The reduction of Cr(VI) to Cr(III) was followed by the since the recirculation provided complete mixing in the
formation of sediments, which caused obstruction of the flow bioreactor and therefore spatial homogeneity. In samples
along the filter depth (partial or complete pore clogging) and taken from the effluent of the reactor the Cr(VI) concen-
consequently insufficient exploitation of the entire filter vol- tration at the end of each recirculation cycle was found
ume. In order to overcome sediment formation and make well below the permitted limit of Cr(VI) for drinking water
better use of the entire filter, we switched the filter to sequenc- (0.05 mg/l).
ing batch reactor operating mode with recirculation. The filter
was loaded with nutrients and hexavalent chromium solution
to a final concentration of 30 mg/l. After several operating
cycles the period was reduced to only 40 min (Fig. 5).
Fig. 6 shows the reduction of hexavalent chromium and
the consumption of sodium acetate during an operating cycle
of 40 min. It is obvious that sodium acetate is in excess
(390 mg/l as organic carbon) while only about 120 mg/l of
organic carbon is necessary for the reduction of about 33 mg/l
of hexavalent chromium.
A reduction of the organic carbon to 265 mg/l did not alter
the duration of the operating cycle, which remained at 40 min
(Fig. 7a). Further reduction of the organic carbon to about
150 mg/l reduced even further the duration of the operating
cycle to only 30 min (Fig. 7b), while for lower organic carbon
concentrations down to 100 mg/l the duration of the operating
cycles remained constant at 30 min. Reduction of the organic
carbon concentration to about 20 mg/l, led to carbon source
limitation as shown in Fig. 7c. From this figure it is apparent
that the bulk sodium acetate concentration is consumed in the
first 20 min while slow desorption of organic carbon from the
biofilm structure leads to very slow reduction of the remaining Fig. 6. Hexavalent chromium reduction and sodium acetate consumption
hexavalent chromium. during an operating cycle of 40 min (SBR operation with recirculation).
 E. Dermou et al. / Journal of Hazardous Materials B126 (2005) 78–85 83

Fig. 7. Operating cycles of SBR operation with recirculation for various sodium acetate concentrations: (a) 265 mg C/l; 40 min, (b) 150 mg C/l; 30 min, (c)
20 mg C/l; 340 min.

3.3. Continuous operation 4. Discussion

Finally, the continuous operating mode was tested. At present the most commonly used technology for
This operating mode (gravity flow) ensures fully aerobic treatment of heavy metals in wastewater is chemical precip-
conditions without the need for any external aeration source. itation. Conventional chemical treatment involves reduction
However, the use of the continuous operating mode did not of Cr(VI) to Cr(III) by reducing agent under low pH (2–3)
improve filter performance. The maximum chromate removal conditions and subsequent adjustment of solution pH to
rate (200 g Cr(VI)/m2 d) was achieved for a volumetric flow near neutral ranges to precipitate Cr(III) as hydroxides [5].
rate of 30 ml/min resulting in a retention time of only 1.3 min, Thus, chemical Cr(VI) reduction requires energy input and
while for higher flow rates chromate removal rate was large quantities of chemicals. Biological reduction could,
less. therefore, provide a useful alternative economical process
84 E. Dermou et al. / Journal of Hazardous Materials B126 (2005) 78–85

[20]. However, until now, the biological chromium reduction form seems to be a promising solution. In this work a new
was tested only in small laboratory devices, in suspension approach for biological hexavalent chromium reduction was
processes, and using pure cultures. Recently, attempts using introduced. The use of indigenous bacterial populations
attached growth processes appeared in the literature with provides a certain advantage and ensures durability under
promising results. This is the first time it was attempted various operating conditions. Trickling filters proved very
to attack the serious industrial environmental problem of promising devices providing a support material for consistent
hexavalent chromium disposal, using mixed cultures and a biofilm structure development while minimizing operating
pilot-scale attached growth process bioreactor. cost since physical aeration is adequate for bacterial needs.
The inability of maintaining pure cultures under industrial SBR operation with recirculation proved to be a very
conditions made it necessary to look for mixed cultures effective operating mode, since it ensures even wetting of
able to overcome industrial scale difficulties and possible the filter and distribution of the precipitates all over filter
contamination. As discussed earlier, we searched for volume. The high removal rates of hexavalent chromium that
indigenous bacterial population in a contaminated industrial were achieved indicate a feasible, economical and efficient
sludge while the dominant species in the culture we used technique for biological hexavalent chromium removal from
was found to be Acinetobacter sp. It is worth noting that industrial wastewater effluents.
we did not use aseptic (sterilized) conditions while at the
same time we used tap water and sodium acetate as carbon
source. The operating concentration of 30 mg/l of hexavalent Acknowledgments
chromium that was used, is extremely high, since in the
common Hellenic Aerospace Industry untreated effluents is This work was supported by the Hellenic Aerospace
about only 5 mg Cr(VI)/l. On the other hand concentrations Industry S.A. The authors wish to thank Prof. K. Bourtzis
up to 120 mg/l did not seem to be toxic for the bacterial and S. Siozios for their help with the identification of bacte-
culture we used, indicating that the specific culture appears rial strains.
to be resistant in an actual industrial environment.
The use of an attached growth system provides the
necessary surface for the development of biofilm structures.
Biofilms provide high biomass concentration per unit vol- References
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