Metal Bioremediation Through Growing Cells PDF

Environment International 30 (2004) 261 – 278
www.elsevier.com/locate/envint
Metal bioremediation through growing cells

Anushree Malik *
Department of Applied Chemistry, Faculty of Engineering, Utsunomiya University, 7-1-2 Yoto, Utsunomiya, Tochigi 321-8585, Japan
Received 1 May 2003; accepted 4 August 2003
Abstract
Heavy-metal pollution represents an important environmental problem due to the toxic effects of metals, and their accumulation
throughout the food chain leads to serious ecological and health problems. Metal remediation through common physico-chemical techniques
is expensive and unsuitable in case of voluminous effluents containing complexing organic matter and low metal contamination.
Biotechnological approaches that are designed to cover such niches have, therefore, received great deal of attention in the recent years.
Biosorption studies involving low-cost and often dead/pretreated biomass have dominated the literature and, subsequently, extensive reviews
focusing on equilibrium and kinetics of metal biosorption have also come up. However, the low binding capacity of biomass for certain
recalcitrant metals such as Ni and failure to effectively remove metals from real industrial effluents due to presence of organic or inorganic
ligands limit this approach. At times, when pure biosorptive metal removal is not feasible, application of a judicious consortium of growing
metal-resistant cells can ensure better removal through a combination of bioprecipitation, biosorption and continuous metabolic uptake of
metals after physical adsorption. Such approach may lead to simultaneous removal of toxic metals, organic loads and other inorganic
impurities, as well as allow optimization through development of resistant species. However, sensitivity of living cells to extremes of pH or
high metal concentration and need to furnish metabolic energy are some of the major constraints of employing growing cells for
bioremediation. The efforts to meet such challenges via isolation of metal-resistant bacterial/fungal strains and exploitation of organic wastes
as carbon substrates have began. Recent studies show that the strains (bacteria, yeast and fungi) isolated from contaminated sites possess
excellent capability of metal scavenging. Some bacterial strains possess high tolerance to various metals and may be potential candidates for
their simultaneous removal from wastes. Evidently, the stage has already been set for the application of metal-resistant growing microbial
cells for metal harvesting. This review focuses on the applicability of growing bacterial/fungal/algal cells for metal removal and the efforts
directed towards cell/process development to make this option technically/economically viable for the comprehensive treatment of metal-rich
effluents.
D 2003 Elsevier Ltd. All rights reserved.
Keywords: Metal; Growing cells; Bioremediation; Metal resistance
1. Introduction known quantities of pollutants in the water they consume.

The main sources of heavy-metal pollution are mining,
Industrialization has long been accepted as a hallmark milling and surface finishing industries, discharging a
of civilization. However, the fact remains that industrial variety of toxic metals such as Cd, Cu, Ni, Co, Zn and
emanations have been adversely affecting the environment. Pb into the environment. In the last few decades, concen-
Industrial effluents containing toxics and heavy metals tration of these heavy metals in river water/sediments has
drain into the river, which is often the source of drinking been clearly demonstrated. Eventually, build-up of danger-
water for another town downstream. Municipal water ous concentrations of toxic metals in grains and vegetables
treatment facilities in most of the developing countries, grown in contaminated soils is most alarming due to
at present, are not equipped to remove traces of heavy harmful effects of metals on human health. It is well
metals, consequently exposing every consumer to unknown that heavy metals can be extremely toxic as they
damage nerves, liver and bones, and also block functional
groups of vital enzymes (Moore, 1990; Ewan and Pam-
* Tel.: +81-28-689-6168; fax: +81-28-689-6167.
phlett, 1996). Some of the metals like Ni are also listed as
E-mail addresses: anumalik@cc.utsunomiya-u.ac.jp, a possible human carcinogen (group 2B) and associated
anushree_malik@yahoo.com (A. Malik). with reproductive problems and birth defects. Besides, a
0160-4120/$ - see front matter D 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envint.2003.08.001
262 A. Malik / Environment International 30 (2004) 261–278
range of detrimental effects on fauna and flora are also sorption studies involving low-cost, easily available, dead or
well documented. Often, these contaminants also inhibit live biomass have dominated the literature (Mogollon et al.,
biological remediation processes due to metal sensitivity of 1998; Singh et al., 2001). Metal uptake capacity by various
the strain and necessitate additional combat strategies for biosorbents (algae/fungi/yeasts) has been evaluated using
efficient operation (Malik, 2000; Malik et al., 2001). Since biosorption isotherm curves derived from equilibrium batch
these heavy metals are a valuable resource for different sorption experiments and effect of various process param-
industrial applications, their recovery and recycle assumes eters such as pH, biomass loading, biomass pretreatments,
even greater significance. Further, strict environmental etc. have been studied extensively (Sag et al., 2000; Yu and
regulations compel industries to shift to cleaner production Kaewsarn, 2000). Further, desorption of adsorbed metals
methods, demanding the development of environmental using dilute eluents and cyclic use of regenerated biomass
friendly, low-cost and efficient treatment technique for has also been demonstrated (Chang et al., 1997). However,
metal rich effluents. recently, it was reported that live Aspergillus niger cells
Although the removal of toxic heavy metals from indus- possessed higher Ni biosorption capacity than dead biomass
trial wastewaters has been practiced for several decades, the pretreated with sodium hydroxide, detergents, formaldehyde
cost-effectiveness of the most common physico-chemical and dimethyl suphoxide, probably due to intracellular Ni
processes such as oxidation and reduction, chemical precip- uptake (Kapoor et al., 1999). Similar reports indicating
itation, filtration, electrochemical treatment, evaporation, significant metal uptake by growing cells of various micro-
ion-exchange and reverse osmosis processes is limited. High organisms call for reviewing the performance of such
reagent requirement and unpredictable metal ion removal processes in order to carve a niche in the scenario dominated
are some other disadvantages associated with such techni- by purely biosorptive processes.
ques. Further, strong and contaminating reagents are used
for desorption, resulting in toxic sludge and secondary
environmental pollution. These disadvantages can become 2. Why growing cells?
more pronounced and further aggravate the process cost in
case of contaminated ground waters, mine tailings effluent Gadd (1988) and Brierley (1990) have described the
and other industrial wastewaters due to voluminous efflu- many ways in which bacteria, fungi and algae can take up
ents containing complexing organic matter and low metal toxic metal ions. Heavy-metal ions can be entrapped in the
contamination. Biotechnological approaches can succeed in cellular structure and subsequently biosorbed onto the
those areas and are designed to cover such niches. Micro- binding sites present in the cellular structure. This method
organisms have evolved various measures to respond to of uptake is independent of the biological metabolic cycle
heavy-metal stress via processes such as transport across the and is known as ‘‘biosorption’’ or ‘‘passive uptake’’. The
cell membrane, biosorption to cell walls and entrapment in heavy metal can also pass into the cell across the cell
extracellular capsules, precipitation, complexation and oxi- membrane through the cell metabolic cycle. This mode of
dation– reduction reactions (Rai et al., 1981; Macaskie and metal uptake is referred to as ‘‘active uptake’’. The metal
Dean, 1989; Huang et al., 1990; Avery and Tobin, 1993; uptake by both active and passive modes can be termed as
Brady and Duncan, 1994; Brady et al., 1994; Krauter et al., ‘‘bioaccumulation’’. Most of the studies dealing with
1996; Veglio et al., 1997). They have proven capability to microbial metal remediation via growing cells describe
take up heavy metals from aqueous solutions, especially the biphasic uptake of metals, i.e., initial rapid phase of
when the metal concentrations in the effluent range from biosorption followed by slower, metabolism-dependent
less than 1 to about 20 mg/l (Brierley, 1990). Besides, active uptake of metals (Garnham et al., 1992; Donmez
flexibility to handle the range of physico-chemical param- and Aksu, 1999). Recent reports employing growing
eters in effluents, selectivity to remove only the desired cultures of marine microalgae indicate that intracellular
metals and the cost-effectiveness are some added advan- Cd levels are often higher than the biosorbed ones (Mat-
tages of biological metal cleanup techniques. These factors sunaga et al., 1999; Perez-Rama et al., 2002). Mostly
have promoted extensive research on the biological methods during the studies employing harvested biomass (dead/
of metal removal. pretreated), metal is not taken up into the cells; rather, it
High metal-binding capacities of several biological mate- is just adsorbed at the cell surface (Torres et al., 1998) and,
rials have already been identified in part. Among the thus, only a small fraction of bioaccumulation capacity is
biosorbents, there are marine algae (Volesky and Holan, exploited.
1995), bacteria (Hartmeier and Berends, 1995), yeasts Although high biosorptive potential for several types of
(Sugawara et al., 1997), fungi and waste mycelia from the dead/pretreated microbial biomass has been reported, their
fermentation (Luef et al., 1991) and food industry (Senthil- strength in field remains to be tested. Most of the metal
kumar et al., 2000). Further, the capacities of these micro- biosorption studies are conducted using synthetic metal
organisms to accumulate an ample range of metal species solutions (Ahuja et al., 1999; Mehta and Gaur, 2001), and
have also been described (Volesky, 1994; Fourest et al., when the biosorption potential using real industrial effluent
1994; Volesky and Holan, 1995). In the recent past, bio- is tested, the efficiency turns out to be very low. Corder and
A. Malik / Environment International 30 (2004) 261–278 263
Reeves (1994) found improvement in Ni biosorption (from strain selection and exploitation of organic wastes as carbon
NiCl2 solution in deionized water) capacity by autoclaving substrates. The isolation and selection of metal-resistant
of cyanobacteria biomass. However, none of the three strains shall be a crucial aspect to overcome the prime
species could bind Ni efficiently in actual effluent sample constraint of employing living cell systems. Incidentally,
due to the presence of high concentration of sodium ions. resistant cells are expected to bind substantially more
Thus, current methods of biosorption are sensitive to ambi- metals, which in turn is a prerequisite for enhanced bio-
ent conditions such as pH, ionic strength and the presence of precipitation/intracellular accumulation and development of
organic or inorganic ligands. Moreover, biosorption also an efficient process. Instead of depending upon single
lacks specificity in metal binding (Baudet et al., 1988). species, a better approach could be towards designing a
Some metals like Ni are more recalcitrant pollutant, and consortium of strains having high metal biosorption, bio-
many microorganisms have a relatively low Ni-binding accumulation and bioprecipitation capacities (Pumpel et al.,
capacity (Basnakova and Macaskie, 1977; Chan et al., 2001). Multispecies consortia can better withstand extreme
1991). As per the reports, pure Ni sorption to dead biomass conditions often encountered with industrial wastewater
only reaches a loading of 0.05 –0.3% at 2– 5 mg/l concen- like spikes of pH or high metal concentration. The rich
tration of Ni (Savvaidis et al., 1992; Kutsal, 1995; Scott et exopolymer content of the biofilms may also be beneficial
al., 1995; Tsezos et al., 1995). This shall result in large for both entrapping dispersed solids and biosorption of
volumes of sludge. Reusability of biomass after desorption dissolved metals. Further, they provide a microenvironment
is possible only if relatively weak chemicals are used for (like alkaline pH, high concentrations of CO2), which could
desorption. Thus, the resulting eluents are low in concen- be very beneficial for metal precipitation. The positive
tration of metals and pose problem in final recovery of interaction between constituent species may also facilitate
metals. Further, biomass needs to be exchanged after a the survival of sensitive strains (Bradshaw et al., 1998). In
maximum of 5 – 10 sorption – desorption cycles. Due to view of these facts, the applicability of growing cells in
these facts, it seems to be impossible to develop a contin- suitably configured bioreactor appears to offer promising
uous system based only on biosorptive removal of metals biotechnology for combating heavy-metal pollution in the
using microbial biomass. environment.
As discussed above, many a times, biosorption alone Several studies on application of growing microbial
may not suffice for effective metal remediation. Under such cells for metal scavenging have been reported. However,
situation, application of active and growing cells might be a in toxic metal removal applications, it is important to
better option due to their ability of self-replenishment, ensure that the growing cells can maintain a constant
continuous metabolic uptake of metals after physical ad- removal capacity after multiple bioaccumulation– desorp-
sorption, and the potential for optimization through devel- tion cycles, and a suitable method is required to optimize
opment of resistant species and cell surface modification the essential operating conditions. The situation demands a
(Wilde and Benemann, 1993; Sandau et al., 1996). Further, multi-prong approach including strain isolation, cell devel-
the metals diffused into the cells during detoxification get opment and process development in order to make the
bound to intracellular proteins or chelatins before being ultimate process technically and economically viable.
incorporated into vacuoles and other intracellular sites. These issues form the theme of the present review besides
These processes are often irreversible and ensure less risk a brief picture of the mechanisms of accumulation and
of metal releasing back to the environment (Gekeler et al., localization of accumulated metals in living cells. In
1988). Apart from this, using growing cultures in biore- addition, other integrated microbial processes, which some-
moval could avoid the need for a separate biomass produc- how differ from the conventional biosorption, have also
tion process, e.g. cultivation, harvesting, drying, processing been discussed.
and storage prior to the use. In contrast to conventional
chemo-physical and biosorptive methods, employment of
active microorganisms may allow development of a single- 3. Isolation of resistant strains and their efficiency
stage process for removal of most of the pollutants present
in industrial effluents. Growing cells have unlimited capac- Recent studies show that the strains isolated from con-
ities to cleave organo-metallic complexes, degrade organic taminated soils and electroplating effluent-contaminated
compounds, as well as take up other inorganic ions such as sludge have excellent capability of removing significant
ammonium, nitrate and phosphate. Further, dissolved and amounts of metals from both aqueous solution as well as
fine-dispersed metallic elements can also be removed via electroplating effluent. Since there has been a vast range of
immobilization. metal and strain combinations explored in various studies, in
Yet, there are significant practical limitations to bioup- a bid to present them clearly, this discussion is compart-
take by living cell systems such as sensitivity of the system mentalized depending upon the metals used. To facilitate
to extremes of pH, high metal/salt concentration and comparison, various informations (scale of experiment,
requirement of external metabolic energy (Donmez and employed metal concentrations and achieved metal removal
Aksu, 2001). However, such challenges can be met via efficiencies) are tabulated in Tables 1– 3.
3.1. Cadmium optimal strategy for metal remediation. In addition, several

bacterial isolates from effluents of local industries exhibited
Some recent reports on resistance and bioaccumulation resistance to Cr, Cu, Pb, Zn, Cd and Ni; and often multiple
capacity of Cd by isolated microorganisms under growing metal resistance was observed (Nuzhat et al., 2001). Further,
conditions are shown in Table 1. Obviously, bacterial, nature of growth media, pH and temperature of incubation
fungal, as well as algal strains, are potential candidates for affected the metal resistance, indicating optimization of
such applications. Roane and Pepper (2000) observed that operational conditions was possible.
different strains of Cd-resistant bacterial isolates varied in Various strains of filamentous soil fungi isolated from the
their resistance level due to potentially varied mechanism of sediments of industrially polluted streams (Table 1) removed
resistance. Significant reduction of soluble Cd was observed 63– 70% Cd during the 13-day growth period (Massaccesi et
during growth of plasmid-bearing Bacillus strain H9 and al., 2002). An important observation in this study was that
Pseudomonas strain H1. Similarly, three strains of bacteria although more biomass was produced in shaken cultures as
isolated from industrial effluents (Enterobacter cloacae and compared to the static ones, the Cd removal (%) and removal
Klebsiella spp.) were resistant to high concentrations of Cd, efficiency (Cd ppm/g dry weight) were much higher in static
Pb and Cr in the growth media and could remove approx- cultures. Thus, optimum conditions for metal removal may
imately 85% Cd during growth (Haq et al., 1999). Although not be the same as that for maximum growth and biomass
the genes for Cd and Cr resistance were present on plasmid production and in such cases may favor an economic process
DNA, the gene for Pb resistance was shown to be present on due to nonrequirement of agitation. Further, the strains could
chromosomal DNA. Further, the Cd removal efficiency was effectively grow and remove metal at a 100-fold higher Cd
growth-phase-dependent. E. cloacae and Klebsiella strain 1 level than what was present in their natural habitat. The
showed higher efficiency during late log phase, while results indicate that microbial population inhabiting polluted
Klebsiella strain 2 was more efficient in early log phase. sites may have ability to resist much higher concentrations
This study implicated the advantage of plasmid-encoded Cd and thus efforts need to be directed in revealing and
resistance gene, allowing for further genetic manipulation exploiting their real potential.
and enhanced efficiency. Moreover, a continued effort to Growing cultures of marine algae like Tetraselmis suecica
evaluate the metal removal from real effluents with cheap also have high resistance and Cd removal capacity (Perez-
nutritional sources to sustain growth could lead to an Rama et al., 2002). Although Cd was toxic and affected the
Table 1
Microbial resistance and bioaccumulation of Cd by isolated strains
Microorganism Name of organism Resistance Removal Scale of Comment Reference
(isolation source) (MRL)/uptake (%) experiment/source
efficiency of metal
(concentration)
Bacteria Arthrobacter strain D9 50 Ag/ml 22 (48 h) LS (25 ml)/GM Resistance mechanisms Roane and Pepper (2000),
(metal-contaminated Bacillus strain H9 275 Ag/ml 36 (48 h) (3 – 150 mg/l) vary with strain Roane et al. (2001)
soil) Pseudomonas strain H1 225 Ag/ml 36 (48 h)
Bacteria Enterobacter cloacae 220 Ag/ml 86 (24 h) LS/GM Plasmid-based Haq et al. (1999)
(industrial effluents) CMCB-Cd1 (100 mg/l) resistance
Klebsiella spp. 110 Ag/ml 87 (24 h)
CMBL-Cd2
Klebsiella spp. 110 Ag/ml 85 (24 h)
CMBL-Cd3
Fungi (industrially Aspergillus terreus 122 (ppm/g) 70 (13 days) LS (250 ml)/GM Higher Cd removal Massaccesi et al. (2002)
polluted sediments) Cladosporium 21 (ppm/g) 63 (13 days) (10 mg/l) efficiencies in static
cladosporiodes cultures than shaken
Fusarium oxysporum 60 (ppm/g) 63 (13 days) ones
Glicocladium roseum 184 (ppm/g) 65 (13 days)
Penicillum spp. 37 (ppm/g) 60 (13 days)
Talaromyces helicus 150 (ppm/g) 70 (13 days)
Trichoderma koningii 82 (ppm/g) 64 (13 days)
Marine algae Tetraselmis suecica EC50: 59.6 LS (2 l)/GM Removal linked to Perez-Rama et al. (2002)
7.9 Ag/ml (6 days) (0.6 – 45 mg/l) sulfhydril gr.
concentration
Marine algae Chlorella spp. 39.4 mg/g 48.7 LS (100 ml)/GM Matsunaga et al. (1999)
NKG16014 (14 days) (5.6 mg/l)
Bacteria Bacillus subtilis 9.5 M/g – LS/GM Also accumulates Costa et al. (2001)
biomass Pb, Zn, Cu
MRL: maximum resistance level. LS: lab-scale. GM: growth media.
growth of this microalga at 0.6 –45 mg/l concentrations, the Chlorella miniata WW1 for cyclic Ni biosorption showed
total Cd removed by cells increased as its concentration in that high efficiency of 85% removal was maintained for five
the medium was increased. In general, there was a rapid cycles but thereafter progressively declined, reaching 70%
accumulation of Cd in the first 2 days of the 6-day growth removal at the end of 10th cycle (Tam et al., 2001). Cells
period. At higher concentration (45 mg/l), the accumulation were still viable after successive Ni biosorption, but division
continued and a maximum was recorded on the fifth day of rate and chlorophyll a activity were adversely affected.
the culture. Thus, the response of cells to metal stress These strains were much efficient than the live cyanobacte-
depends upon the ambient concentration of metal. rial strains of Anabaena and Nostoc spp. previously tested
(Corder and Reeves, 1994). However, testing of their real
3.2. Nickel, cobalt and vanadium potential to remove Ni from industrial effluents still
remains.
As shown in Table 2, algal strain isolated from domestic Candida spp. isolated from sewage samples could accu-
sewage treatment plant had much higher uptake efficiency mulate significant amount of Ni (57 –71%) and Cu (52 –
for Ni as compared to the commercial strain (Wong et al., 68%), but the process was found to be affected by initial
2000). Moreover, the viability of cells was restored after Ni metal concentration and pH (optimum 3– 5) of the medium
biosorption in case of resistant isolate. Although this study (Donmez and Aksu, 2001). Larger fraction of metal was
focused on rapid biosorption of Ni and did not analyze accumulated during the end of exponential growth phase
intracellular Ni accumulation by growing cells, viability of (9 –15 days), indicating intracellular accumulation and ad-
isolate after exposure to high concentrations of Ni indicates aptation of cells enhanced the resistance and metal ion
that growing cells can also be employed. Repeated use of accumulation. The results indicate that a self-replenishing
Table 2
Microbial resistance and bioaccumulation of Ni, Co and V by isolated strains
Microorganism Name of Metal MRL/uptake Removal Scale of Comment Reference
(isolation source) organism efficiency (%) experiment/source
of metal
(concentration)
Algae (acquired) Chlorella Ni 0.6 mg/g 33 – 41 LS/metal solution Cell viable after Ni Wong
vulgaris (24 h) (10 – 40 mg/l) accumulation but et al. (2000)
growth rate reduced
Waste water Chlorella Ni 1.4 mg/g > 99
treatment plant miniata (24 h)
(WW1)
Cyanobacteria Anabaena Ni 2 mg/g LS/metal Low Ni biosorption Corder and
cylindrica solution or in actual effluents Reeves (1994)
Anabaena Ni 6 mg/g effluent
flos aquae ( < 20 mg/l)
Nostoc spp. Ni 3 mg/g
Yeast (sewage) Candida spp. Ni 10.3 – 46.8 29 – 57 LS (100 ml)/GM Metal concentration Donmez and
(nonadapted) mg/g (5 – 15 days) (66 – 514 mg/l) and pH-dependent Aksu (2001)
Candida spp. Ni 10.9 – 30.8 44 – 71 uptake
(adapted) mg/g (5 – 15 days)
Fungi Aspergillus Ni 0.38 mg/ml 98 (4 days) LS/GM Ni uptake requires Magyarosy
(metal-contaminated niger (23.5 mg/g) (381 mg/l) energy metabolism et al. (2002)
soil)
Bacteria Pseudomonas Ni 74.9 mg/g LS/metal Concentration-dependent Ghozlan
(local isolate) spp. solution uptake et al. (1999)
Bacteria Escherichia Ni 2 mg/ml LS Outer membrane Hernandez
(oil refinery soil) hermannii (10.2 mg/g) (10 ml)/metal profile altered in et al. (1998)
CNB50 V 1 mg/ml solution presence of metals
(53.8 mg/g) 100 mg/l)
Enterobacter Ni 2 mg/ml
cloacae (6.9 mg/g)
CNB60
V 1 mg/ml
(39.3 mg/g)
Fungi Neurospora Co – 90 (24 h) LS/GM Growth medium affects Karna
crassa or metal removal et al. (1996)
solution
(500 mg/l)
LS: lab-scale. GM: growth media.
system employing such strains can be left to run continu- more in live cells. However, Sar et al. (1998) reported that
ously for extended periods. Similarly, a strain of A. niger Ni uptake by P. aeruginosa is affected by growth medium as
isolated from metal-contaminated soil had unusually high the cells grown in minimal medium could remove only 146
resistance (0.38 mg/ml) to Ni, which became more pro- nM Ni/mg protein/h, while those grown in enriched medium
nounced (0.47 mg/ml) when grown on solid medium removed 950 nM Ni/mg protein/h. They attributed this
(Magyarosy et al., 2002). Although it removed >98% Ni enhanced uptake to improved metabolic state of the cells.
under growing conditions, the metal removal by nongrow- Similarly, Co removal (90%) by a Co2 +-resistant Neuros-
ing live cells was very low. Further, inhibition of Ni removal pora crassa cor was growth medium-dependent as the
by addition of protonophore confirmed that Ni removal is mycelia grown in nitrate N-medium were found to be more
not only due to the biosorption, rather an active process efficient than those grown in ammonium N-medium (Karna
requiring energy metabolism. Another study with soil fungi et al., 1996). Thus, it appears that Ni uptake is dependent
(Penicillium italicum 90SL) shows that presence of Ni in upon the metabolic state of cells.
growth medium negatively affected growth rate, but when
Ni was added as shock factor, such negative influence was 3.3. Copper, lead, chromium and zinc
less evident (Levinskaite et al., 2000). Here, the inhibition
of metabolic activity (represented by oxygen consumption) Several yeast strains have been tested for bioaccumula-
was more prominent after 24 h of exposure rather than after tion of Cu under growing conditions (Table 3). The level of
the first 4 h. On the other hand, biosorption study on Ni Cu accumulation and microbial growth were dependent on
involving various fungi reports that Ni accumulation is fast the pH and initial Cu concentration. The results indicate that
and does not require microorganism’s active metabolism Kluveromyces marxianus, Candida spp. and Saccharomyces
(Mogollon et al., 1998). cerevisiae remove 73 – 90% of Cu during their growth
The role of indigenous bacteria in Ni remediation is (Donmez and Aksu, 1999; Donmez and Aksu, 2001),
evident from the fact that a bacteria-populated column although considerable differences in Cu uptake capacity of
(containing carbon carriers) demonstrated higher Ni remov- different strains were found. Authors predicted that meta-
al than the unpopulated column (Grabas and Kolwzan, bolically active cells from the exponential growth phase
2000). The bacteria used for immobilization were isolated probably contain highly active enzymes, some of which
from bottom deposit of a pond, which receives galvanic may be involved in complexing and binding the metal ions.
process wastes. Similarly, magnetite-immobilized cells of Metal ion uptake in yeasts is known to involve an initial
Enterobacter spp. 4-2 could remove 50 – 90% Ni under rapid biosorption of metal ions to negatively charged sites
optimum conditions (Wong and Fung, 1997). A highly on the cell wall (Brady and Duncan, 1994; Krauter et al.,
specific Ni transport system has been identified in bacterial 1996) followed by a slower, energy-dependent entry into the
cells of Helicobacter pyroli (Mobley et al., 1995), and this cell. Both the outer mannan –protein layer of the yeast cell
has been used in construction of metal-resistant strains via wall as well as the inner glucan – chitin layer play important
genetic engineering. Some bacterial strains show multiple- role in heavy-metal accumulation (Avery and Tobin, 1993;
metal resistance; for example, strains (belonging to the Brady et al., 1994). A majority of intracellular metals
family Enterobacteriaceae) isolated from contaminated soil become bound to polyphosphate granules localized in and
at oil refinery have shown accumulation of both Ni and V near the vacuoles or may also get detoxified via binding to
(Hernandez et al., 1998). In addition, Serratia marcescens specific low-molecular weight proteins, namely, metallo-
strain 7 and E. agglomerans strain 16 possessed very high thioneins and phytochelatins (Volesky et al., 1992; Volesky
tolerance to Ni (under living condition at pH 7), with the and May-Philips, 1995). Previous studies indicate absorp-
latter one efficient for both Ni and Co (Gomez et al., 2001). tion of metals by insoluble cellular material as the main
These strains, which were subsequently classified as super- mechanism of metal accumulation in K. marxianus and S.
resistant to Ni, showed a shift in protein patterns under cerevisiae (Yazgan and Ozcengiz, 1994). Viable cells of S.
metal stress. Valentine et al. (1996) also isolated a filamen- cerevisiae were even able to remove metals (Cu, Cr, Cd, Ni
tous bacterium from a depth of 96.2 m (Bacillus simplex and Zn) from electroplating effluents, and their performance
ZAN-044), and their strain showed a rapid, pH-dependent was further enhanced by glucose pretreatment of cells (Stoll
binding of Cd, Ni and Co. Such strains may be potential and Duncan, 1996). Another Cu-resistant strain (S. cerevi-
candidates for simultaneous removal of various metals from siae SN41) was found to successfully remove Cu from wine
wastes. during fermentation (Brandolini et al., 2002).
There had been conflicting reports on whether cell Fungal and bacterial strains are also not behind in the
metabolism is essential for Ni uptake. Strains of Pseudo- race (Table 3). A. niger could remove significant quantities
monas spp. (P. fluorescens 4F39, P. stutzeri) have often been of Cu and Pb from growth media but was less resistant
used for biosorption of Ni (Lopez et al., 2000; Ramteke, against Cr (Dursun et al., 2003). The bioaccumulation
2000). Ghozlan et al. (1999) reported that both live as well capacity as well as the optimal pH for growth in presence
as dead cells of locale isolate of Pseudomonas spp. had of metals was different in case of Cu and Pb. Recently,
similar Ni uptake capacity, although Co and Cd uptake was efficient Zn uptake by growing cells of Aspergillus spp.
Table 3
Bioaccumulation of Cu, Pb and Cr by growing/live cells
Microorganism Name of Metal MRL/Uptake Removal Scale of Comment Reference
(isolation source) organism efficiency (%) experiment/metal
source
(concentration)
Yeast (sewage) Candida spp. Cu 4.7 – 23 mg/g 22 – 52 LS (100 ml)/GM Removal depends Donmez and
(nonadapted) (8 days) (100 – 1500 mg/l) upon metal Aksu (2001)
Candida spp. Cu 5.3 – 37 mg/g 5 – 68 concentration
(adapted) (8 – 13 days) and pH
Yeast (acquired) Saccharomyces Cu 2 – 9 mg/g 13 – 74 LS (100 ml)/GM Removal depends Donmez and
cerevisiae (4 days) (50 – 700 mg/l) upon metal Aksu (1999)
Kluveromyces Cu 1.8 – 11.2 mg/g 10 – 90 concentration
marxianus (4 days) and pH
Schizosaccharomyces Cu 0.6 – 1.3 mg/g 11 – 25
pombe (4 days)
Candida spp. Cu 1.6 – 14.8 mg/g 13 – 73
(4 days)
Fungi (acquired) Aspergillus niger Cu 4.9 – 15.6 mg/g 19 – 57 LS (100 ml)/GM Optimum pH for Dursun
(7 days) (25 – 150 mg/l) growth and metal et al. (2003)
Pb 5.3 – 34.4 mg/g 13 – 88 (25 – 500 mg/l) accumulation is
(7 days) metal-dependent
Cr 5.1 – 6.6 mg/g 21 – 36 (25 – 75 mg/l)
(7 days)
Bacteria Pseudomonas Cr 0.08 mg/ml 46 LS (20 ml)/GM Variable effect on Hassen
(wastewater) aeruginosa (2 days) (69 mg/l) growth, protein et al. (1998)
Bacteria Bacillus Cu 0.03 mg/ml 34 (18 mg/l) synthesis and
(acquired) thuringiensis (2 days) pigment production
Bacteria Thiobacillus Cu 0.02 mg/ml 25 LS (1.5 l)/metal Live cells more Boyer
(adapted) ferrooxidans (700 mg/g) (15 min) solution (1000 mg/l) efficient et al. (1998)
Yeast Sacchaomyces Cu 0.32 mg/ml 90 LS (100 ml)/grape Cu removal Brandolini
(culture cerevisiae SN41 (7 days) must (320 mg/l) from wine et al. (2002)
collection)
Bacteria Pseudomonas Pb 0.5 mg/ml 80 LS (100 ml)/metal Uptake pH and Chang
aeruginosa PU21 (110 mg/g) (2 days) solution (100 mg/l) growth et al. (1997)
(Rip64) Cu 0.3 mg/ml 75 (20 mg/l) phase-dependent
(23 mg/g) (2 days)
LS: lab-scale; GM: growth media.
isolated from industrial waste has been reported (Sharma et metabolism-dependent or a surface phenomenon. Table 4
al., 2000; Sharma et al., 2002). This process could be shows the chemical nature and site of accumulated Ni in
successfully employed for complete removal of Zn from different systems. In case of A. niger, Ni was found to be
real industrial effluents (containing 46 mg Zn/l) in a lab- associated with cell wall as well as inside the cell (Mag-
scale continuous flow system at 10 g/l sugar concentration yarosy et al., 2002). The chemical nature was analyzed by
(Sharma et al., 2003). Several bacterial strains also show X-ray and electron diffraction analysis to conclude that Ni
Cu/Cr uptake (Table 3), although their growth and metab- accumulated as nickel oxalate dehydrate crystals. Most of
olism is severely affected under metal stress. Cu-resistant the other studies involving bacterial and fungal strains
Pseudomonas putida strain S4 isolated from smelter drain- indicated that Ni was primarily restricted to cell surface or
age of copper mines could not only accumulate metals from to periplasm and cell membrane (Table 4). However, Maier
metal-supplemented growth medium but also removed Cu et al. (1990) previously reported that Ni accumulation in
and Zn from mine effluents, low-grade ore and ore tailings Bradyrhizobium japonicum was intracellular and related to
(Saxena et al., 2001; Choudhury and Srivastava, 2002). the Ni-binding soluble proteins. Interesting results on the
mechanism of Cd resistance in bacterial isolates were
revealed by the work of Roane et al. (2001). Of the four
4. Localization of bioaccumulated metals in cell isolates, Pseudomonas strain H1 and Bacillus strain H9,
which were resistant to higher concentration (225 and 275
In the last few decades, with the advent of sophisticated Ag/ml), appeared to use an intracellular mechanism of Cd
techniques (Tables 4 and 5) facilitating deeper insight into sequestration. The resistance mechanisms of these two
the cell structure, many studies have focused on the local- organisms were linked to plasmid-encoded genes. Although
ization of accumulated metals inside cell. Eventually, one the exact mechanism of intracellular accumulation could not
can also predict through these results, whether the uptake is be elucidated, metallothionein production and polyphos-
phate precipitation could be the two possible explanations. The studies with growing algal cells confirm that a higher
The two other Cd-resistant isolates (20 and 50 Ag/ml), proportion of accumulated Cd is intracellular, suggesting an
Pseudomonas strain I1a and Arthrobacter strain D9, did internal detoxification mechanism (Table 4). As a common
not carry plasmids but showed evidence of EPS (extracel- response to Cd, microalgae are considered to synthesize
lular polysaccharides) production upon staining. Transmis- intracellular metal-binding peptides (Class III metallothio-
sion electron microscopy (TEM) of these strains showed Cd neins) as Cd binds to the –SH groups of these molecules
accumulation external to the cells. Thus, the resistance during detoxification (Kaplan et al., 1995). Report confirm-
mechanism and subsequently the location of accumulated ing increased class III metallothioneins in cultures exposed
metal vary with the strain. Similarly, Roane (1999) reported to Cd stress (6 mg/l) and binding of accumulated Cd to this
that the degree and mechanism of Pb resistance for two group has also appeared (Perez-Rama et al., 2001). Recent-
bacterial isolates corresponded with their environmental Pb ly, Perez-Rama et al. (2002) found a positive correlation
exposure. P. marginalis, isolated from a soil contaminated between sulfhydryl groups and intracellular Cd in living
with high total (but low soluble) Pb showed higher resis- cells of T. suecica. After 6 days of growth, the ratio of
tance and extracellular Pb exclusion with high amount of sulfhydryl groups and intracellular Cd was 2 at all the Cd
EPS production. On the other hand, B. megaterium isolated concentrations tested. The study indicates that although the
from soil containing high soluble Pb showed lower resis- cells show no growth at very high concentration of Cd (45
tance and intracellular accumulation of Pb. This strain mg/l), the cells are alive and are synthesizing higher
produced no discernable EPS as reportedly observed by amounts of sufhydryl groups to tolerate the toxic affect of
polarization microscopy. Cd. Under such conditions, cells can silently continue
Table 4
Localization and chemical nature of bioaccumulated Ni and Cd
Organism Metal Localization Chemical nature Techniques Comment Reference
of metal employed
Aspergillus niger Ni Cell wall Rectangular Analytical electron Energy metabolism Magyarosy
and inside crystals of nickel microscopy (AEM), essential et al. (2002)
cell oxalate dehydrate energy dispersive
X-ray spectroscopy
(EDXS),
X-ray and electron
diffraction analysis
(XRD)
Pseudomonas Ni 88% restricted Nickel phosphide Transmission Potential role of Sar et al.
aeruginosa to periplasm and (Ni5P4, NiP2, Ni12P5), electron microscopy phosphoryl and (2001)
cell membrane nickel carbide (TEM), carboxyl/carbonyl gr.
(Ni3C) crystals EDXS, XRD of cell wall/membrane
Pseudomonas Ni Cell surface Needle and TEM, EDXS, XRD Rapid and Lopez et al.
fluorescens 4F39 hexagon-like ppt pH-dependent (2000)
of Ni (OH)2 accumulation
at pH 9
Rhizopus spp. Ni Cell surface TEM Rapid and energy Mogollon et al.
metabolism not (1998)
required
Bradyrhizobium Ni Intracellular Ni-binding Nonexchangable Maier et al.
japonicum soluble proteins accumulation (1990)
Pseudomonas spp. Cd Extracellular TEM, EDXS No plasmid Roane et al.
strain I1a (EPS) (MRL 20Ag/ml) (2001)
Pseudomonas spp. Cd Intracellular Plasmid
strain H1 (cytoplasmic) (MRL 225 Ag/ml)
Bacillus spp. strain H9 Cd Intracellular Plasmid
(cytoplasmic) (MRL 275 Ag/ml)
Tetraselmis suecica Cd Intracellular (67%), Metal analysis in Correlated to Perez-Rama
cell surface (33%) different fractions sulphuhydryl gr. et al. (2002)
concentration
Chlorella spp. Cd Intracellular (67%), Metal analysis in Maximum surface Matsunaga
NKG16014 cell surface (25%), different fractions adsorption in et al. (1999)
ppt (4%), 2 days but
unknown (4%) intracellular
accumulation until
8 days
EPS: extracellular polysaccharide.
accumulating Cd intracellularly without showing the appar- reported depending upon the strain and the metal concerned
ent growth and diverting all the energy towards metal (Tables 4 and 5). Tsezos et al. (1997) observed the bio-
detoxification. This facet of live and growing cells to sorption sites of metals using various microbial strains (BP
metabolically respond to high metal concentration has no 7/26 Arthrobacter spp., ER 121 Alkaligenes eutrophus and
counterpart in pure biosorption processes using dead/treated AS302 P. mendocina) and metals (Pd, Ag, Y and Ni). The
biomass. localization of the biosorbed metal appeared to be metal-
Using a combination of instrumental techniques, Torres dependent rather than strain-dependent. In P. aeruginosa,
et al. (1998) confirmed the role of polyphosphate bodies enhanced accumulation of Cu is linked to higher amount of
(PPB) in accumulation of Zn, Pb, Mn and Al in Plectonema EPS production (Kazy et al., 2002). Presence of Cu ions in
boryanum. They observed that living cells with active the growth medium caused stimulation of four-fold EPS
uptake system are more efficient in sequestering of metals production in Cu resistant (Cur) strain while such response
through PPB. Suh et al. (1998), using TEM, observed that was not exhibited by the sensitive (Cus) strain. Cu2 +-
the accumulated Pb gradually enters the S. cerevisiae cell, binding capacity of the EPS of Cur was also greater (320
and most of it gets deposited in cytoplasm after 2 h. They mg/g) than the EPS of Cus (270 mg/g). While studies with
inferred a three-step mechanism comprising metabolism- B. japonicum showed that the lipololysaccharide (LPS) and
independent first step (3– 5 min) when Pb binds to cell wall, not the EPS is responsible for metal (Cd, Cu, Pb, Zn)
followed by metabolism-dependent second step (5 min –24 binding (Oh et al., 2002). LPS mutant (lacking O-polysac-
h) in which Pb accumulated on cell wall/membrane and, charide part) bound 50 –70% lower concentration of metals
finally, third step of Pb accumulation in cytoplasm (after 24 than the wild strain, although its EPS composition was
h). The study could not ascertain whether the third step is unaltered. Thus, it appears that LPS molecules of B.
metabolism-dependent or -independent. Previously, White japonicum have properties which effect precipitation of
and Gadd (1986) also reported about intracellular accumu- metal-rich mineral phases. Langley and Beveridge (1999),
lation of Cd, Co and Cu by trained (adapted) cells of S. however, proposed that the negatively charged sites located
cerevisiae, with Co mostly localized to the vacuoles. They in the O-side chains are not directly responsible for the
observed that the mechanisms for accumulation of different binding of metallic ions in P. aeruginosa. However, the B-
metals were distinct and varied in their stability when the band LPS molecule as a whole may contribute to overall cell
strains were detrained in metal-free medium. Thus, it surface properties, which favor the precipitation of distinct
appears that in case of yeast and microalgae as well, most metal-rich mineral phases.
of the metals are accumulated intracellularly. From the reports discussed above, it appears that varied
The story is different in case of bacterial strains as both mechanisms of metal accumulation result in different local-
extracellular exclusion and intracellular accumulation are ization of the accumulated product. These variations in the
Table 5
Localization of bioaccumulated heavy metals
Organism Metal Localization Techniques employed Comment Reference
Pseudomonas Pb Extracellular (EPS) TEM, EDXS High MRL (2.5 mM) Roane (1999)
marginalis and more EPS production
Bacillus Pb Intracellular (cytoplasm) Low MRL (0.6 mM)
megaterium and negligible EPS
Saccharomyces Pb Cell wall and membrane TEM three-step mechanism of Suh et al. (1998)
cerevisiae (after 3 min), cytoplasm accumulation indicated
(after 2 h)
Pseudomonas Cu Potentially EPS GC and FTIR for Cur strain produces more Kazy et al. (2002)
aeruginosa EPS composition EPS; purified EPS binds Cu
Pseudomonas Ag More intracellular SDS-PAGE of purified Optimum accumulation in Ibrahim et al. (2001)
diminuta (Ag-binding proteins) Ag-binding proteins early exponential phase
Saccharomyces Co Vacuole TEM Training of strains alters White and Gadd (1986)
cerevisiae Cd Soluble fraction intracellular distribution
Cu Soluble fraction
Bradyrhizobium Cd, Cu, Lipopolysaccharide SDS-PAGE of LPS rather than EPS has Oh et al. (2002)
japonicum Pb, Zn (LPS) isolated LPS metal binding property; LPS
mutant binds less metals
Plectonema Zn, Pb, Polyphosphate bodies TEM, EDXS PPB in living cells with active Torres et al. (1998)
boryanum Mn, Al (PPB) uptake system remove more
metal
Chlorella salina Co, Mn, Zn Intracellular – Metabolism-dependent second Garnham et al. (1992)
(higher concentration uptake phase (slow)
in vacuole)
GC: gas chromatography. FTIR: fourier transform infrared spectroscopy. SDS-PAGE: sodium dodecylsulfate-polyacrylamide gel electrophoresis.
mechanism arise out of the toxicity of the metal concerned could grow well in presence of higher metal concentration
as well as the environmental conditions to which the while the nonadapted ones perished. Moreover, the specific
microbial strain was exposed. In general, short biosorption metal uptake capacity and the metal removal (%) by adapted
studies have low possibility to observe and appreciate the cells were higher than the nonadapted cells at all the
delayed intracellular accumulation and hence, most of such concentrations tested. Although the authors could not eluci-
studies conclude with the surface adsorption of the metal. date the mechanism of adaptation, they implied constitutive
However, the studies, which monitor metal removal by synthesis of metallothionein/other copper-binding proteins
growing cells often realize the metabolically linked intra- or changes in the genetic make up as one of the factors in
cellular accumulation. Recent studies have proved that in adaptation. Further, in growing cell systems, availability of
spite of low apparent growth, growing cells are able to enough energy reserves facilitates active transport of metals
remove metals continuously through internal detoxification for deposition into the vacuoles. Similarly, in case of bacteria
mechanisms. Application of growing cells in bioremediation also, more tolerant strains have been produced via adapta-
can well exploit these facts. However, there remains a great tion. Baillet et al. (1997) adapted Thiobacillus ferrooxidans
challenge to be faced via further development of the strain strain via successive exposure to higher concentrations of
and the process. Cd. The biomass (living but nongrowing) of adapted strain
had Cd2 + uptake capacity of 0.31 g/g dry weight as com-
pared to 0.21 g/g dry weight in case of nonadapted cells. On
5. Cell development the other hand, although Cu tolerance of a standard strain of
T. ferrooxidans could be enhanced from 0.3 to 0.6 M, the
As discussed above, microorganisms have evolved var- specific Cu2 + uptake capacity of adapted strain reduced to 0.
ious mechanisms of metal resistance and scientists have 09 from 0.7 g/g dry weight in nonadapted cells (Boyer et al.,
tried to exploit genetic/metabolic basis of all such mecha- 1998). Co- and I-tolerant cyanobacterial strains (Spirulina
nisms for production of superior strains. Although chromo- platensis) have also been produced and shown to be more
somal genes may also be involved, bacterial resistance to efficient than the parent strain for intracellular uptake of
heavy metals is often conferred by products of genes these contaminants (Singh and Kumar, 1994). The LD50
situated on plasmids (Silver et al., 2001), rendering genetic value for Co-adapted strain was about 2.5 times higher than
manipulation for strain improvement easy and feasible. that of the parent strain.
Often, multiple-metal resistance is associated with the same There are some nonconventional approaches to strain
plasmid (Nuzhat et al., 2001). In addition, several successful selection. Although S. cerevisiae is often used for metal
attempts to clone eukaryotic MTs (Metallothioneins) in bioremediation, recently reported comparison of a flocculent
bacteria for the cytoplasmic production and then for surface and nonflocculent strain for Cu2 + removal appears to be a
expression have been recently reviewed (Valls and Lorenzo, novel and interesting approach (Soares et al., 2002). Lab-
2002) and shall not be dealt here in detail. Cytoplasmic scale bioaccumulation experiments were conducted in 500-
expression of metal-binding moieties has been coupled with ml plastic flasks containing 200 ml MES buffer (pH 6.0, 0.2
introduction of specific heavy-metal transporters (Chen and mM Cu) and 0.4 mg/ml cell concentration. Flocculent strain
Wilson, 1997). Krishnaswamy and Wilson (2002) con- S646-1B accumulated more Cu2 + (81 nM/mg dry weight)
structed a genetically engineered E. coli strain by introduc- than the nonflocculent S646-8D strain (30 nM/mg dry
ing nixA gene (coding for Ni transport system) from H. weight) in the first 10 min of contact with the metal. The
pyroli into JM 109 cells, which expressed a glutathione S- authors envisage this to the presence of additional metal-
transferase – pea metallothionein fusion protein. This strain binding sites on cell surface of flocculent strains. Floccula-
was capable of accumulating threefold higher Ni as com- tion of yeasts is supposed to be mediated by lectin –
pared to cells expressing MT without the transporter. Such saccharide like interactions. During this process, Ca2 + is
approach, however, is limited to only those metals for which required, which probably ensures the correct conformation
active transport system is known. Cytoplasmic expression of of the lectins. It is possible that other divalent cations like
metal-binding polypeptides like phytochelatins (Robinson et Cu2 + may also play the same role, thereby enhancing the
al., 2001), possible engineering of LPS and EPS in gram- metal-binding capacity of flocculent strain. Moreover, other
negative bacteria (Langley and Beveridge, 1999) and design divalent metals such as Zn2 +, Cu2 + and Ni2 + also induced
of novel metal-binding peptides (Sousa et al., 1996; Bae et rapid flocculation of this strain. The use of flocculent strains
al., 2000; Hong et al., 2000) are some other approaches for metal remediation offers several advantages due to
currently being pursued. natural sedimentation of the cells after waste treatment such
Another much practiced way of producing more resistant as design simplicity, low energy costs and avoidance of cell
and efficient strain is through adaptation of the cells to immobilization. Efforts have been made to construct cell
progressively higher concentrations of heavy metals. Don- surface-engineered yeast, which can bind and aggregate in
mez and Aksu (2001) adapted strains of Candida species response to Cu ion (Kuroda et al., 2002). Although self-
(isolated from sewage) to Cu and Ni by serial subcultures in flocculating sewage bacteria are famous for bioflocculation
Cu- and Ni-supplemented growth medium. Adapted cells process, coaggregation among dominant nonflocculating
sludge bacteria was recently revealed (Malik et al., 2002). almost similar after each cycle, indicating that biofilm
While the mechanism of such highly specific intergeneric efficiency was constantly maintained even after multiple
coaggregations is pair-dependent, potential role of lectin – desorption with dilute HCl (Costley and Wallis, 2001).
saccharide interactions is visible in few pairs (Malik and The biofilm comprising yeast, bacteria and filamentous
Kakii, 2003a,b; Malik et al., 2003). Multispecies consortia organisms could withstand short-term disruptions and
are considered more efficient over the monospecies culture soon recovered from the shock. Even higher strength of
due to greater resistance against environmental fluctuations desorbing HCl (0.5 M) facilitating rapid desorption did
and the metabolic relations among the member strains. not reportedly damage the biofilm. These encouraging
Evaluation of the metal removal capabilities of such coag- results shall direct more efforts towards application of
gregates and their stability under heavy-metal stress may be growing mixed consortia in suitably configured reactors
very interesting. for metal removal. However, as the metallic effluents are
often nutrient limiting, they must be supplemented to
support microbial growth. Biotechnological approaches
6. Process development for removal of low metal contaminated voluminous
effluents can be economically viable only if cheap carbon
After development and selection of a suitable strain, and nutrient sources replace the conventional substrates
the next step is that of the process development. Several used in lab studies. In a bid to reduce energy cost, use of
studies have reported improvements in metal removal by molasses has been shown to support good growth and Cu
immobilization of yeast, algal or bacterial cells (Duncan bioaccumulation property of K. marxianus (Aksu and
et al., 1995; Yusef, 1997; Ghozlan et al., 1999). Using a Donmez, 2000). Using a small-scale set up (100 ml),
lab-scale, up-flow algal column reactor packed with 75 concentration of molasses sucrose was optimized (20 g/l),
ml alginate –algal beads, 97% Cu and 91% Ni could be which lead to 48.4% removal of Cu in 8 days. Another
removed from synthetic solutions (30 ml/l Cu or Ni) with aspect that aggravates the cost is the choice of support
a HRT (Hydraulic Retention Time) of 30 min (Lau et al., material. Cheap, locally available support materials for
1988). It has been found that Ni sorption level by development of metal-removing bacterial biofilms are also
immobilized bacterial biomass depends upon the surface being explored (Nuzhat et al., 2001).
type and the structure of the carbon carrier pores (Grabas Use of moving bed sand filters (effective bed height, 2
and Kolwzan, 2000). Efforts have been made to optimize m) for development of metal reducing biofilm via inocu-
the immobilization conditions for maximum metal remov- lation of a consortium of resistant, metal biosorbing/bio-
al. Wong and Fung (1997) reported that under optimum precipitating bacteria has been demonstrated (Diels et al.,
immobilization conditions (cell/magnetite ratio of 1:10, 2001a). Although nutrients (3.4 mg NO3-N/l) and carbon
pH 6.0 and temperature 25 jC) more than 90% cells of substrate (8 mg carbon/l) need to be supplied to maintain
Enterobacter spp. 4-2 could be immobilized on support, growth, this novel approach results in significant removal
leading to very high Ni2 + removal efficiency. Most of (0.8 mg/l) of Ni as well as other inorganic and organic
these studies aim at biosorption using nongrowing cells. pollutants. The process termed as MERESAFIN (MEtal
However, process developments employing growing mi- REmoval by SAnd Filter INoculation) was also successful
crobial cells, which are more relevant to this review, have at pilot scale for treatment of rinsing waters from nickel
also come up. plating line to reduce Ni in the effluent (Diels et al., 1999;
Pumpel et al., 2001). The mixed bacterial population
6.1. Reactor design and operational strategy termed as five-mix comprised of P. mendocina AS302,
Arthrobacter spp. BP7/26, Ralstonia eutropha CH34, P.
A set of studies demonstrated feasibility of employing fluorescens and Methylobacillus spp. MB 127. Results of
rotating biological contactor (RBC), supporting the immo- the preliminary batch studies found very high Ni removal
bilized growing biofilms for removal of Cu, Zn and Cd and the Ni-load of biomass was estimated to be about 50
from synthetic wastewater. The inoculum consisted of the mg/g dry weight, which exceeded the simple Ni biosorp-
enrichment cultures out of the sewage activated sludge tion capacity (2.7 – 3.5 mg/g dry weight) of individual
and the nutrient broth concentration had been judiciously strains. This shows that other biologically mediated pro-
chosen to avoid metal complexation. The disc rotation cesses such as bioprecipitation play important role in Ni
speed (10 rev/min) and flow rate (6.9 ml/min) had to be remediation. Further, growing bacterial cells are able to
previously optimized for maximum biofilm growth and cleave the complexes of nickel with organic acids, which
metal removal (Costley and Wallis, 1999, 2000). This are also subsequently degraded, while the released Ni is
laboratory scale RBC (10-l working volume) used nutri- biosorbed and largely bioprecipitated. This study demon-
ent broth as carbon source and was run for multiple strated feasibility of cheap carbon source for growth of
sorption (84 days) and desorption (48 h) cycles with a selected bacterial strains and based on the process cost,
HRT of 24 h. Average metal removal for Cu (81.8%), Zn calculations concluded that such a process will be techni-
(49.7%) and Cu (30.1%) after 84 days of contact was cally as well as economically viable.
Acidophilic chemolithotrophs including T. ferrooxidans hollow fiber membrane bioreactor system for the treatment
are also known for high metal tolerance. Using a bioelec- of AMD using hydrogen consuming SRB biofilms (Tabak
trochemical reactor for regeneration of substrate, a 6.4-fold and Govind, 2003). Authors suggest that the membrane
increase in biomass of T. ferrooxidans DSM583 was ob- bioreactor is advantageous over the conventional bioreactor
served in 55 h growth period together with 20-h electrolysis. due to large microporous membrane surface, prevention of
The generated biomass could be efficiently used for metal H2S mixing with pressurized hydrogen gas inside the
removal from synthetic solutions as well as industrial membrane, prevention of washout problems due to biofilm
effluents (Magnin et al., 1998). Another patented system formation and lower operating costs.
termed BMSR (Bio Metal Sludge Reactor) uses stirred tank A major limitation of active bioremediation of AMD is
reactors fed with contaminated soil, water and nutrients sensitivity of SRB to acidity and high metal concentration
(Diels et al., 2001b). R. metallidurans CH34 that is used for requiring minimization of contact between the H2S-pro-
metal removal also improves the settling of sludge, so that ducing bioreactors and acidic wastewaters by employing
cells and the treated sludge are easily separated in a settling two-stage reactors (USEPA Annual Report, 2001). How-
device. This process could reduce the Cd concentration from ever, investigations have lead to the isolation (from mine
9 to 1 mg/kg dry soil. sites) of pure and mixed cultures of SRB that are active at
low pH and resistant to high metal concentrations so that a
6.2. Technologies for metal recovery and treatment of acid direct on-line sulfidogenic systems can be used, whereby
mine drainage (AMD) the AMD (a source of sulfate as well as heavy metals) is
fed directly into a SRB bioreactor (Hard et al., 1997;
A major source of the metal pollution is metal-rich acid Kolmert and Johnson, 2001). Injection of nutrients to
mine drainage (AMD) and acid rock drainage (ARD) from stimulate activity of indigenous SRB, which appears to
waste rock piles. Highly acidic metal-bearing wastewater be the most feasible alternative to treat abandoned under-
generated by the oxidation of metal sulfides to sulfates by ground mines or waste dumps has also been demonstrated
bacteria (Acidithiobacillus ferrooxidans) at active or aban- on field-scale (Lee and Saunders, 2003). Metal-contami-
doned mining sites, is of concern due to toxic effect on flora nated groundwater (from car battery recycling plant) con-
and fauna. Lab-scale studies to control iron-oxidizing bac- tains Pb, Cd, Zn and Cu under prevailing acidic and
teria in AMD by optimizing growth of heterotrophic bac- aerobic conditions. Indigenous bacteria (primarily Desulfo-
teria such as Acidiphilium acidophilum have been reported sporosinus orientis) assist in sulfate reduction and produ-
(Marchand and Silverstein, 2000; Marchand and Silverstein, ces favorable conditions (low Eh and high pH), resulting
2002). The precipitation of metal sulfides utilizing hydro- in almost complete precipitation of metal sulfides as
gen sulfide produced by sulfate-reducing bacteria (SRB) mineral phases (Saunders et al., 2001). Integrated Passive
has received more attention (Sen and Johnson, 1999). The Biological Treatment Process uses a series of biological
Mine Waste Technology Program (MWTP) of the United processes (involving both anaerobic and aerobic bacteria)
States Environmental Protection Agency (USEPA) and for the comprehensive treatment of AMD (Table 6). The
Department of Energy (DOE) has lead to several field scale attractive features of this technology such as use of
demonstrations of such bioremediation technologies (Table inexpensive wastes as nutrient source, elimination of
6). Passive treatment systems (using wetlands or compost power requirement and constant attention make it espe-
reactors) are designed to optimize processes that occur in a cially suitable for treatment of underground and abandoned
natural wetland with the main focus on either aerobic or sites.
anaerobic processes (MEND Report 3.14.1, 1996). Never-
theless, this alternative has an important drawback as the 6.3. Integrations
precipitated metals are retained (in the organic matrix)
rather than recovered, and their long-term fate is often An integration of acidophilic bioleaching (using sulfur-
unsure. oxidizing bacteria) with subsequent bioprecipitation of the
Active treatment methods often use SRB maintained in leached metals as insoluble sulfides by sulfate reducing
separate bioreactors for optimum H2S production, which is bacteria has been reported (White et al., 1998). About 69%
then fed into tanks containing metal-rich wastewater, caus- of the toxic metals (Cu, Ni, Mn) could be leached in 175
ing rapid precipitation and recovery of metal sulfides. days, and 80 –98% of the leached metals could be precip-
Several SRB-based bioremediation processes have been itated as metal sulfides in next step by the activity of
demonstrated as pilot-scale operations (Table 6) and have sulfate-reducing bacteria in an anaerobic bioreactor. Such
further proceeded to industrial applications. However, future an approach although produced effluents that qualified
scale-up challenges to meet seasonal temperature and metal environmental criterion, seems limited by the slow rate of
load variations, and to design for the progressive biological first (bioleaching) step. Another hybrid approach to Ni
degradation of the variable (cheap and locally available) remediation has been termed as microbially enhanced
organic substrates still remain (Gusek, 2003). Recent study chemisorption of heavy metals (MCHEM). This is based
reports about development of an innovative polypropylene on uranyl ion accumulation by a Citrobacter spp. as
Table 6
Biological treatment systems for recovery of metals and control of AMD
Technology Site Scale/reactor design Organism Target Removal (%); Comment Reference
influent/flow metal concentration
rate in treated effluent
A. Malik / Environment International 30 (2004) 261–278

Passive Wetland United Keno Pilot-scale (29.5 60.6 ft, Carex Zn (30 mg/l) 80; 5000 Ag/ml Optimization needed United Keno
Treatment System Hill Mine site surface area, 1790 ft2) aquatilis containing to reduce Zn to 500 Ag/ml Hill Mine Case
AMD/10 l/s Study, (1995)
Active Treatment Callopie Mine, Pilot-scale, 3 SRBR AMD/1 – 3 gpm Zn 500 Ag/ml Mn, Fe, Al, As also Zaluski et al., (2001);
Butte, MT RII 71.5 ft, below ground SRB HRT 5.5 days Cu 5 Ag/ml removed; no effect of USEPA, (2002)
RIII 61 ft, below ground SRB HRT 4.5 days Cd 80 Ag/ml winter freeze on R IV
RIV 72.5 ft, above ground SRB HRT 5.5 days performance
Integrated Passive Surething Mine, Field scale four stage system SRB, Metal (Al, Fe, Al 99.7, Cd 97.1, No need of power USEPA (2001)
Biological Treatment Southwest Montana Primary Anaerobic Reactor aerobic Mn, Zn) rich Cu 99.6, Fe 99, and frequent attention;
Process # bacteria AMD Pb 75.4, Mn 86.3, uses cow manure and
Passive Alkalinity Addition Zn 97.2, As 82.8 walnut shells as C source
#
Secondary Anaerobic Reactor
#
Aerobic Reactor ðFe=Mn removalÞ
Selective Sequential Berkeley Pit, MT Bench-scale and pilot-scale SRB AMD High purity metal ppt Extensive details on Tabak and
Precipitation (SSP) six-stage SSP with hollow and re usable water reactor oper. and removal Govind, (2003)
fiber membrane bioreactor that meets USEPA’s data to be presented later
Gold standard at Int. Conference
In-situ treatment Underground Injection of nutrients into SRB Metal-rich Sig. metal precipitation Same as above Lee and
by biostimulation aquifer, Troy, AL underground wastewater effluent from observed Saunders, (2003)
car battery
recycling plant
AMD: acid mine drainage. SBR: sulfate reducing bacteria. SRBR: SRB reactor. HRT: hydraulic retention time.
273
crystals of HUO2PO44H2O (HUP), using enzymatically cadmium-resistant isolates supported the degradation of
generated inorganic phosphate. Cells preloaded with HUP 500 Ag/ml 2,4-D by the cadmium-sensitive 2,4-D degrader
then remove Ni2 + by intercalative ion exchange forming R. eutropha JMP134 in 120 h. Authors observed that 48-
crystals of Ni(UO2PO4)27H2O (Bonthrone et al., 1996). In- h time lap was needed between inoculation of the metal-
depth studies employing scanning transmission electron detoxifying population and the next inoculation with the
microscopy with electron probe X-ray microanalysis organic-degrading population for effective reduction of
(EPXMA) and proton-induced X-ray emission analysis metal and organic contaminant. This study relied on
(PIXE) have been conducted to analyze the molar ratios primary metal detoxification so that organic-degrading
and localization of nickel uranyl phosphate deposits asso- microbial populations were prevented from metal stress
ciated with Citrobacter spp. cells immobilized in polyacryl- and inhibition. The strategy was also found successful at
amide gel (Basnakova et al., 1998). The results confirmed pilot scale with 5-gallon soil bioreactor containing 27 kg
presence of deposits on the cell surface as well as between soil, as significant reductions in 2,4-D levels were
the cells, indicating the role of EPS in the creation of achieved within six weeks.
intercellular deposits.
Bacterial consortium capable of using metal-cyanide as
nitrogen source has been used for biodegradation and detox- 7. Concluding remarks
ification of nickel cyanide present in industrial wastewater
(Patil and Paknikar, 2001). Under optimal conditions, this The examples reviewed above indicate that bioremedi-
consortium could grow in presence of 0.5 mM Ni-cyanide ation using growing microbes is a feasible alternate to pure
and caused its efficient (99%) degradation using sugarcane biosorptive removal of metal contaminants from complex
molasses as the carbon source. In a related lab-scale study (50 industrial effluents. It is evident that the full bioaccumula-
ml), biosorption and biodegradation have been sequentially tion potential of microbes is not realized in short biosorp-
combined to remove metal cyanides. The first step of metal tion processes, which rarely allow intracellular uptake.
cyanide biosorption by Cladosporium cladosporiodes is Isolation of super-resistant strains from contaminated sites
followed by biodegradation of the remaining part by the has, to an extent, eliminated the primary hurdle for appli-
mixed consortium within 5– 6 h (Patil and Paknikar, 1999). cation of growing cells. Genetic engineering may further
enhance the potential of robust environmental strains.
6.4. Dual-augmentation strategies Moreover, a range of reactor designs have been proposed,
some of which have been successful at pilot scale level for
Simultaneous recovery of heavy metals and degradation treatment of real effluents. Use of multiple species consor-
of xenobiotics in a reactor compartmentalized by separa- tia has proved advantageous for higher metal scavenging
tion membrane, which also acts as immobilization support and more stability against environmental fluctuations. In
for bacteria, has been demonstrated (Diels et al., 1995). addition, this approach has also proved to be useful in
Bacteria form a biofilm on the membrane and thus treat biological treatment of other organic wastes via protection
effluent on one side while they are kept active via the rendered by the metal accumulating strains to the organic
nutrient stream on the other side. The reactor is termed as degrading bacteria. Exploitation of locally available sup-
bacteria immobilized composite membrane reactor port material and cheap carbon/nutrition sources for the
(BICMER). Alcaligenes eutrophus CH34 is used for metal cells appears to promise an economically favorable pro-
removal via bioprecipitation, reducing some metals (Cd, cess. Further, ability of growing cells to degrade metal
Zn, Cu, Pb, Y) to less than 50 Ag/kg while others (Co, Ni, complexes and remove other organic/inorganic contami-
Pd, Ge) are reduced to less than 100 Ag/kg. A. eutrophus nants present in the waste may assure an effluent fit for
AE1308 is used for degradation of xenobiotics. Similarly, environmental discharge. However, the choice of consortia
metal accumulating strains in conjunction with R. eutropha and carbon/nutrition source must depend upon the nature of
JMP134 have been employed (Roane et al., 2001) for a effluents due to varied complexing properties of metals and
dual-bioaugmentation strategy to enhance remediation of the nutritional content of the effluent itself. Thus, there can
contaminated soil containing Cd and 2,4-dichlorophenoxy- be no universal process to suit all kinds of metal effluents.
acetic acid (2,4-D). R. eutropha JMP134 alone could not Moreover, successful reproduction of the processes at
degrade 2,4-D in presence of Cd. As discussed earlier, commercial scale as well as the ability of strains to
plasmid-bearing Bacillus strain H9 and Pseudomonas maintain constant metal uptake also need to stand the test
strain H1 are able to reduce significant amount of soluble of time.
Cd (36%) during their growth. Although none of the
cadmium-resistant isolates could degrade 2,4-D in pure
culture (25 ml liquid medium amended with 500 Ag/ml Acknowledgements
2,4-D and 12– 24 Ag/ml Cd) as wells as laboratory soil
microcosms (100 gm soil amended with 500 Ag/ml of 2,4- The Department of Science and Technology (DST),
D and 60 Ag/ml Cd), results showed that each of four Government of India, is gratefully acknowledged.
References Costley SC, Wallis FM. Effect of disc rotational speed on heavy metal
accumulation by rotating biological contactor (RBC) biofilms. Lett
Appl Microbiol 1999;29:401 – 5.
Ahuja P, Gupta R, Saxena RK. Sorption and desorption of cobalt by Os- Costley SC, Wallis FM. Effect of flow rate on heavy metal accumulation by
cillitoria anguistissima. Curr Microbiol 1999;39:49 – 52. rotating biological contactor (RBC) biofilms. J Ind Microbiol Biotech
Aksu Z, Donmez G. The use of molasses in copper(II) containing waste- 2000;24:244 – 50.
waters: effect on growth and copper(II) bioaccumulation roperties of Costley SC, Wallis FM. Bioremediation of heavy metals in a synthetic
Kluveromyces marxianus. Process Biochem 2000; 36(5):451 – 8. wastewater using a rotating biological contactor. Water Res 2001;
Avery SV, Tobin JM. Mechanism of adsorption of hard and soft metal ions 15:3715 – 23.
to Saccharomyces cerevisiae and influence of hard and soft anions. Diels L, Roy SV, Somers K, Willems I, Doyen W, Mergeay M, et al. The
Appl Environ Microbiol 1993;59:2851 – 6. use of bacteria immobilized in tubular membrane reactors for heavy
Bae W, Chen W, Mulchandani R, Mehra RK. Enhanced bioaccumulation of metal recovery and degradation of chlorinated aromatics. J Membr
heavy metals by bacterial cells displaying synthetic phytochelatins. Sci 1995;100(3):249 – 58.
Biotechnol Bioeng 2000;70:518 – 24. Diels L, Spaans PH, Van Roy S, Hooyberghs L, Wouters H, Walter E, et al.
Baillet F, Magnin JP, Cheruy A, Ozil P. Cadmium tolerance and uptake by a Heavy metals removal by sand filters inoculated with metal sorbing and
Thiobacillus ferrooxidans biomass. Environ Technol 1997; 18:631 – 8. precipitating bacteria. In: Amils R, Ballester A, editors. Biohydrome-
Basnakova G, Macaskie LE. Microbially enhanced chemisorption of nickel tallurgy and the environment toward the mining of the 21st century: Part
into biologically synthesized hydrogen uranyl phosphate: a novel sys- B. Molecular biology, biosorption, bioremediation. Amsterdam: Elsev-
tem for the removal and recovery of metals from aqueous solutions. ier; 1999. p. 607 – 16.
Biotechnol Bioeng 1977;54:319 – 28. Diels L, Spaans PH, Van Roy S, Hooyberghs L, Wouters H, Walter E, et al.
Basnakova G, Spencer AJ, Palsgard E, Grime GW, Macaskie LE. Identi- Heavy metals removal by sand filters inoculated with metal sorbing and
fication of the nickel uranyl phosphate deposits on Citrobacter sp. cells precipitating bacteria. Process Metall 2001a;11B:317 – 26.
by electron microscopy with electron probe X-ray microanalysis and by Diels L, De Smet M, Hooyberghs L, Kinnaer L, Brox G. Bioremediation of
proton-induced X-ray emission analysis. Environ Sci Technol 1998; heavy metal contaminated soils with a biometal sludge reactor (BMSR).
32(6):760 – 5. Process Metall 2001b;11B:479 – 86.
Baudet C, Sprott GD, Patel GB. Adsorption and nickel uptake in Metha- Donmez G, Aksu Z. The effect of copper(II) ions on growth and
nothrix concilii. Arch Microbiol 1988;150:338 – 42. bioaccumulation properties of some yeasts. Process Biochem 1999;35:
Bonthrone KM, Basnakova G, Lin F, Macaskie LE. Bioaccumulation of 135 – 42.
nickel by intercalation into polycrystalline hydrogen uranyl phosphate Donmez G, Aksu Z. Bioaccumulation of copper(II) and nickel(II) by the
deposited via an enzymic mechanism. Nat Biotechnol 1996;14(5): non-adapted and adapted growing Candida spp. Water Res 2001;
635 – 8. 35(6):1425 – 34.
Boyer A, Magnin J-P, Ozil P. Copper ion removal by Thiobacillus ferroox- Duncan JR, Brady D, Rose PD. The use of immobilized yeast cells for
idans biomass. Biotechnol Lett 1998;20(2):187 – 90. heavy metal removal from wastewaters. In: Holmes DS, Smith RW,
Bradshaw DJ, Marsh PD, Watson GK, Allison C. Role of Fusobacterium editors. Miner bioprocess II. Proc Eng Found Conf Minerals. Warren-
nucleatum and coaggregation in anaerobe survival in planktonic and dale, PA: Metals and Materials Society; 1995. p. 189 – 97.
biofilm oral microbial communities during aeration. Infect Immun Dursun AY, Ulsu G, Cuci Y, Aksu Z. Bioaccumulation of copper(II),
1998;66(10):4729 – 32. lead(II) and chromium(VI) by growing Aspergillus niger. Process. Bio-
Brady D, Duncan JR. Chemical and enzymatic extraction of heavy metal chem. 2003;38(10):1647 – 51.
binding polymers from isolated cell walls of Saccharomyces cerevisiae. Ewan KB, Pamphlett R. Increased inorganic mercury in spinal motor neu-
Biotechnol Bioeng 1994;44:297 – 302. rons following chelating agents. Neurotoxicology 1996;17:343 – 9.
Brady D, Stoll AD, Starke L, Duncan JR. Bioaccumulation of metal cations Fourest E, Canal C, Roux JC. Improvement of heavy metal biosorption by
by Saccharomyces cerevisiae. Appl Microbiol Biotechnol 1994; mycelial dead biomasses (Rhizopus arrhizus, Mucor miehei and Pen-
41:149 – 54. icillium chrysogenum): pH control and cationic activation. FEMS Mi-
Brandolini V, Tedeschi P, Capece A, Maietti A, Mazzotta D, Salzano G, et crobiol Rev 1994;14:325 – 32.
al. Saccharomyces cerevisiae wine strains differing in copper resistance Gadd GM. Accumulation of metal by microorganisms and algae. In:
exhibit different capability to reduce copper content in wine. World J Rehm H, editor. Biotechnology: a complete treatise, vol. 6B, special
Microbiol Biotechnol 2002;18:499 – 503. microbial processes vol. 4. Verlagsgesellschaft, Weinheim: VCH;
Brierley CL. Bioremediation of metal-contaminated surface and ground- 1988. p. 401 – 30.
water. Geomicrobiol J 1990;8:201 – 23. Garnham GW, Codd GA, Gadd GM. Kinetics of uptake and intracellular
Chan SS, Chow H, Wong MH. Microalgae as bioabsorbents for treating location of cobalt, manganese and zinc in the estuarine green alga
mixture of electroplating and sewage effluent. Biomed Environ Sci Chlorella salina. Appl Microbiol Biotechnol 1992;37:270 – 6.
1991;4:250 – 61. Gekeler W, Grill E, Winacker EL, Zenk MH. Algae sequester heavy metals
Chang JO, Law R, Chang CC. Biosorption of lead, copper and cadmium by via synthesis of phytochelatin complex. Arch Microbiol 1988;50:
biomass of Pseudomonas aeruginosa PU21. Water Res 1997;31: 197 – 202.
1651 – 8. Ghozlan HA, Sabry SA, Amer RA. Bioaccumulation of nickel, cobalt, and
Chen S, Wilson DB. Construction and characterization of Escherichia coli cadmium by free and immobilized cells of Pseudomonas spp. Fresenius
genetically engineered for bioremediation of Hg(2+)-contaminated envi- Environ Bull 1999;8(7/8):428 – 35.
ronments. Appl Environ Microbiol 1997;63:2442 – 5. Gomez Y, Coto O, Hernandez C, Marrero J, Abin L. Biosorption of nickel,
Choudhury R, Srivastava S. Zinc accumulative properties of Pseudomonas cobalt and zinc by Serratia marcescens strain 7 and Enterobacter ag-
putida strain S4. Asian J Microbiol Biotechnol Environ Sci 2002; glomerans strain 16. Process Metall 2001;11B:247 – 55.
4(2):197 – 202. Grabas K, Kolwzan B. Biostimulation of nickel sorption on carbon carriers.
Corder SL, Reeves M. Biosorption of nickel in complex aqueous waste Inz Ochr Srodowiska 2000;3(3 – 4):333 – 41.
streams by cyanobacteria. Appl Biochem Biotechnol 1994;45/ Gusek JJ. Design challenges for large-scale sulfate reducing bioreactors.
46:847 – 59. To be presented at the Annual International Conference on Soils,
Costa AD, Carlos A, Pereira F. Bioaccumulation of copper, zinc, cadmium Sediments and Water. University of Massachusetts, Amherst, October
and lead by Bacillus sp., Bacillus cereus, Bacillus sphaericus and Ba- 20 – 23; 2003. http://www.umasssoils.com/abstracts2003/Tuesday/
cillus subtilis. Braz J Microbiol 2001;32(1):1 – 5. acidminedrainage.htm.
Haq R, Zaidi SK, Shakoori AR. Cadmium resistant Enterobacter cloacae sorption of nickel and other heavy metals by Pseudomonas fluorescens
and Klebsiella sp. isolated from industrial effluents and their possible 4F39. J Ind Microbiol Biotechnol 2000;24(2):146 – 51.
role in cadmium detoxification. World J Microbiol Biotechnol 1999;15: Luef E, Prey T, Kubicek CP. Biosorption of zinc by fungal mycelial wastes.
283 – 90. Appl Microbiol Biotechnol 1991;34:688 – 92.
Hard BC, Friedrich S, Babel W. Bioremediation of acide mine water using Macaskie LE, Dean ACR. Microbial metabolism, desolubilisation and dep-
facultatively methylotrophic metal-tolerant sulfate-reducing bacteria. osition of heavy metals: metal uptake by immobilized cells and appli-
Microbiol Res 1997;152:65 – 73. cation to the detoxification of liquid wastes. Adv Biotechnol Process
Hartmeier W, Berends A. Biosorption of heavy metals by Bacillus amylo- 1989;12:159 – 72.
liquefaciens: contribution of cell walls. Med Fac Landbouww Univ Magnin J-P, Baillet F, Boyer A, Zlatev R, Luca M, Cheruy A, et al. Enhance-
Gent 1995;60(4b):2585 – 8. ment, by electrochemical substrate regeneration, of the production of a
Hassen A, Saidi N, Cherif M, Boudabous A. Effects of heavy metals on biomass (Thiobacillus ferrooxidans DSM 583) for a biological metal
Pseudomonas aeruginosa and Bacillus thrungiensis. Bioresour Technol removing process. Can J Chem Eng 1998;76(6):978 – 84.
1998;65:73 – 82. Magyarosy A, Laidlaw RD, Kilaas R, Echer C, Clark DS, Keasling JD.
Hernandez A, Mellado RP, Martinez JL. Metal accumulation and vanadi- Nickel accumulation and nickel oxalate precipitation by Aspergillus
um-induced multidrug resisitance by environmental isolates of Escher- niger. Appl Microbiol Biotechnol 2002;59(2 – 3):382 – 8.
ichia hermannii and Enterobacter cloacae. Appl Environ Microbiol Maier RJ, Pihl TD, Stults L, Sray W. Nickel accumulation and storage in Bra-
1998;64(11):4317 – 20. dyrhizobium japonicum. Appl Environ Microbiol 1990;56:1905 – 11.
Hong SH, Gohya M, Ono H, Murakami H, Yamashita M, Hirayama N, Malik A, 2000. Studies on biodesulfurization of coal. PhD thesis. India:
et al. Molecular design of novel metal-binding oligomeric human Indian Institute of Technology Delhi; 2000.
metallothioneins. Appl Microbiol Biotechnol 2000;54:84 – 9. Malik A, Kakii K. Intergeneric coaggregations among Oligotropha carbox-
Huang C, Huang C, Morehart AL. The removal of copper from dilute idovorans and Acinetobacter species present in activated sludge. FEMS
aqueous solutions by Saccharomyces cerevisiae. Water Res 1990; 24: Microbiol Lett 2003a;224:23 – 8.
433 – 9. Malik A, Kakii K. Pair-dependent coaggregation behavior of non-floccu-
Ibrahim Z, Ahmad WA, Baba AB. Bioaccumulation of silver and the iso- lating sludge bacteria. Biotechnol Lett 2003b;25:981 – 6.
lation of metal-binding protein from P. diminuta. Braz Arch Biol Tech- Malik A, Dastidar MG, Roychoudhury PK. Biodesulfurization of coal:
nol 2001;44(3):223 – 5. effect of pulse feeding and leachate recycle. Enzyme Microb Technol
Kaplan D, Heimer YM, Abeliovich A, Goldsbrough PB. Cadmium toxicity 2001;28:49 – 56.
and resistance in Chlorella spp. Plant Sci 1995;109:129 – 37. Malik A, Sakamoto M, Kakii K. Coaggregation of Microbacterium ester-
Kapoor A, Viraraghavan T, Roy Cullimore D. Removal of heavy met- aromaticum S51 with other strains of non-flocculating sludge bacteria.
als using the fungus Aspergillus niger. Bioresour Technol 1999; IWA’s Water Environ Manage Ser 2002:737 – 48.
70:95 – 104. Malik A, Sakamoto M, Ono T, Kakii K. Coaggregation between Aci-
Karna RR, Sajani LS, Maruthi P. Bioaccumulation and biosorption of Co2 + netobacter johnsonii S35 and Microbacterium esteraromaticum strains
by Neurospora crassa. Biotechnol Lett 1996;18(10):1205 – 8. isolated from sewage activated sludge. J. Biosci. Bioeng. 2003;91(1):
Kazy SK, Sar P, Singh SP, Sen AK, D’Souza SF. Extracellular polysac- 10 – 5.
charides of a copper-sensitive and a copper-resistant Pseudomonas aer- Marchand EA, Silverstein J. Remediation of acid rock drainage by induc-
uginosa strain: synthesis, chemical nature and copper binding. World J ing biological iron reduction. Proceedings from the Fifth International
Microbiol Biotechnol 2002;18:583 – 8. Conference on Acid Rock Drainage (ICARD), Denver, CO. Littleton,
Kolmert A, Johnson DB. Remediation of acidic waste waters using immo- CO: The Society for Mining, Metallurgy, and Exploration Inc.; 2000.
bilized, acidophilic sulfate-reducing bacteria. J Chem Technol Biotech- p. 1201 – 7.
nol 2001;76:836 – 43. Marchand EA, Silverstein J. Influence of heterotrophic microbial growth
Krauter P, Martinelli R, Williams K, Martins S. Removal of Cr(VI) from on biological oxidation of pyrite. Environ Sci Technol 2002;36:
ground water by Saccharomyces cerevisiae. Biodegradation 1996; 5483 – 90.
7:277 – 86. Massaccesi G, Romero MC, Cazau MC, Bucsinszky AM. Cadmium re-
Krishnaswamy R, Wilson DB. Construction and characterization of an moval capacities of filamentous soil fungi isolated from industrially
Escherichia coli strain genetically engineered for Ni(II) bioaccumula- polluted sediments, in La Plata (Argentina). World J Microbiol Biotech-
tion. Appl Environ Microbiol 2000;66:5383 – 6. nol 2002;18:817 – 20.
Kuroda K, Ueda M, Shibasaki S, Tanaka A. Cell surface-engineered yeast Matsunaga T, Takeyama H, Nakao T, Yamazawa A. Screening of micro-
with ability to bind, and self-aggregate in response to copper ion. Appl algae for bioremediation of cadmium polluted seawater. J Biotechnol
Microbiol Biotechnol 2002;59(2 – 3):259 – 64. 1999;70:33 – 8.
Kutsal YST. Biosorption of heavy metals by Zoogloea ramigera: use of Mehta SK, Gaur JP. Characterization and optimization of Ni and Cu sorption
adsorption isotherms and a comparison of biosorption characteristics. from aquatic solution by Chlorella vulgaris. Ecol Eng 2001;18:1 – 13.
Chem Eng J 1995;60:181 – 8. MEND Report 3.14.1. Review of passive systems for treatment of acid mine
Langley S, Beveridge TJ. Effect of O-side-chain-lipopolysaccharide drainage. http://www.nrcan.gc.ca/mms/canmet-mtb/mmsl-lmsm/mend/
chemistry on metal binding. Appl Environ Microbiol 1999;65(2): reports/3141es_e.htm; 1996.
489 – 98. Mobley HL, Garner RM, Bauerfeind P. Helicobacter pylori nickel-transport
Lau A, Wong YS, Tong Z, Tam NFY. Metal removal studied by a labo- gene nixA: synthesis of catalytically active urease in Escherichia coli
ratory scale immobilized microalgal reactor. J Environ Sci (China) independent of growth conditions. Mol Microbiol 1995;16(1): 97 – 109.
1988;10(4):474 – 8. Mogollon L, Rodriguez R, Larrota W, Ramirez N, Torres R. Biosorption of
Lee M-K, Saunders JA. Field demonstration and numerical evaluation nickel using filamentous fungi. Appl Biochem Biotechnol 1998;70 –
of in situ bioremediation of metals-contaminated groundwater. To 72:593 – 601.
be presented at the Annual International Conference on Soils, Sedi- Moore JW. Inorganic contaminants of surface water residuals and monitor-
ments and Water. University of Massachusetts, Amherst, October ing priorities. New York: Springer-Verlag; 1990. p. 178 – 210.
20 – 23; 2003. http://www.umasssoils.com/abstracts2003/Tuesday/ Nuzhat A, Uzma B, Raihan S. Resistance and accumulation of heavy
acidminedrainage.htm. metals by indigenous bacteria: bioremediation. In: Nuzhat A, Qure-
Levinskaite L, Rubikas J, Lugauskas A. The effect of nickel on soil fungi shi FM, Khan O, Khan Y, editors. Industrial and environmental
from the genus Penicillium. Ekologia 2000;3:3 – 8. biotechnology. Wymondham, UK: Horizon Scientific Press; 2001.
Lopez A, Larao N, Priergo JM, Marques AM. Effect of pH on the bio- p. 81 – 102.
Oh ET, Yun HS, Heo T-R, Koh S-C, Oh K-H, So J-S. Involvement of fungus. Proc. IlChE Cong, Calcutta, India, vol. 2. Calcutta: Indian
lipopolysaccharide of Bradyrhizobium japonicum in metal binding. J Institute of Chemical Engineers; December 2000. p. 113 – 115.
Microbiol Biotechnol 2002;12(2):296 – 300. Sharma S, Dastidar MG, Sreekrishnan TR. Zinc uptake by a fungal biomass
Patil YB, Paknikar KM. Removal and recovery of metal cyanides using a isolated from industrial wastewaters. ASCE: Practice Period Toxic Haz-
combination of biosorption and biodegradation processes. Biotechnol ard Radioact Waste Manage 2002;6(4):256 – 61.
Lett 1999;21(10):913 – 9. Sharma S, Dastidar MG, Sreekrishnan TR. Biological removal of zinc from
Patil YB, Paknikar KM. Biological detoxification of nickel-cyanide from wastewater using Aspergillus spp. Eur J Miner Process Environ Protect
industrial effluents. Process Metall 2001;11B:391 – 9. 2003;3(1):1 – 4.
Perez-Rama M, Herrero LC, Abalde AJ, Torres E. Class III metallothio- Silver S, Phung LT, Lo J-F, Gupta A. Toxic metal resistances: molec-
neins in response to cadmium toxicity in the marine microalga Tetra- ular biology and the potential for bioremediation. In: Nuzhat A,
selmis suecica (Kylin). Butch Environ Toxicol Chem 2001;20(9): Qureshi FM, Khan O, Khan Y, editors. Industrial and environmental
2061 – 6. biotechnology. Wymondham, UK: Horizon Scientific Press; 2001.
Perez-Rama M, Alonoso JA, Lopez CH, Vaamonde ET. Cadmium removal p. 33 – 41.
by living cells of the marine microalgae Tetraselmis suecica. Bioresour Singh Y, Kumar HD. Adaptation of a strain of Spirulina platensis to grow
Technol 2002;84:265 – 70. in cobalt- and iodine-enriched media. J Appl Bacteriol 1994;76(2):
Pumpel T, Ebner C, Pernfu BB, Schinner F, Diels L, Keszthelyi Z, et al. 149 – 54.
Treatment of rinsing water from electroless nickel plating with a Singh S, Rai BN, Rai LC. Ni(II) and Cr(VI) sorption kinetics by Micro-
biologically active moving-bed sand filter. Hydrometallurgy 2001; cystis in single and multimetallic system. Process Biochem 2001;36:
383 – 93. 1205 – 13.
Rai LC, Gaur JP, Kumar HD. Phycology and heavy metal pollution. Biol Soares EV, Geoffrey DC, Duarte F, Soares HMVM. Use of Sacchromyces
Rev 1981;56:99 – 151. cerevisiae for Cu2 + removal from solution: the advantages of using a
Ramteke PW. Biosorption of nickel(II) by Pseudomonas stutzeri. J Environ flocculent strain. Biotechnol Lett 2002;24:663 – 6.
Biol 2000;21(3):219 – 21. Sousa S, Cebolla A, Lorenzo VD. Enhanced metalloadsorption of bacterial
Roane TM. Lead resistance in two bacterial isolates from heavy metal cells displaying poly-His peptides. Nat Biotechnol 1996;14:1017 – 20.
contaminated soils. Microb Ecol 1999;37:218 – 24. Stoll A, Duncan JR. Enhanced heavy metal removal from wastewater by
Roane TM, Pepper IL. Microbial responses to environmentally toxic cad- viable, glucose pretreated Sacchromyces cerevisiae cells. Biotechnol
mium. Microb Ecol 2000;38:358 – 64. Lett 1996;18(10):1209 – 12.
Roane TM, Josephson KL, Pepper IL. Dual-bioaugmentation strategy to Sugawara H, Yoda A, Kitamoto K, Yamasaki M. Isolation and character-
enhance remediation of cocontaminated soil. Appl Environ Microbiol ization of nickel-accumulating yeasts. Appl Microbiol Biotechnol
2001;67(7):3208 – 15. 1997;48:373 – 8.
Robinson NJ, Whitehall SK, Cavet JS. Microbial metallothioneins. Adv Suh JH, Kim DS, Yun WY, Song SK. Process of Pb2 + accumulation in
Microb Physiol 2001;44:183 – 213. Saccharomyces cerevisiae. Biotechnol Lett 1998;20(2):153 – 6.
Sag Y, Kaya A, Kutsal T. Biosorption of lead(II), nickel(II), and copper(II) Tabak HH, Govind R. Advances in biotreatment of acid mine drainage and
on Rhizopus arrhizus from binary and ternary metal mixtures. Sep Sci biorecovery of metals. To be presented at the Annual International
Technol 2000;35:2601 – 17. Conference on Soils, Sediments and Water. University of Massachu-
Sandau E, Sandau P, Pulz O. Heavy metal sorption by microalgae. Acta setts, Amherst, October 20 – 23; 2003 http://www.umasssoils.com/
Biotechnol 1996;16:227 – 35. abstracts2003/Tuesday/acidminedrainage.htm.
Sar P, Kazy SK, Asthana RK, Singh SP. Nickel uptake by Pseudomo- Tam NFY, Wong JPK, Wong YS. Repeated use of two Chlorella species, C
nas aeruginosa: role of modifying factors. Curr Microbiol 1998;37: vulgaris and WW1 for cyclic nickel biosorption. Environ. Pollut 2001;
306 – 11. 114(1):85 – 92.
Sar P, Kazy SK, Singh SP. Intracellular nickel accumulation by Pseudomo- Torres M, Goldberg J, Jensen TE. Heavy metal uptake by polyphosphate
nas aeruginosa and its chemical nature. Lett Appl Microbiol 2001;32: bodies in living and killed cells of Plectonema boryanum (cyanophy-
257 – 61. cae). Microbios 1998;96(385):141 – 7.
Saunders JA, Lee M-K, Whitmer JM, Thomas RC. In situ bioremediation Tsezos M, Remoudaki E, Angelatou V. A systematic study on equilibrium
of metals-contaminated groundwater using sulfate-reducing bacteria: a and kinetics of biosorptive accumulation: the case of Ag and Ni. Int.
case history. In: Leeson A, editor. Proceedings of international in situ Biodeterior Biodegrad 1995;35:129 – 53.
and on site bioremediation symposium, vol. 9. Columbus, OH: Battelle Tsezos M, Remoudaki E, Angelatou V. Biosorption sites of selected metals
Press; 2001. p. 105 – 12. using electron microscopy. Comp Biochem Physiol., Part A Mol Integr
Savvaidis I, Hughes MN, Poole RK. Differential pulse polarography: a Physiol 1997;118A(3):481 – 7.
method of directly measuring uptake of metal ions by live bacteria United Keno Hill Mine Case Study. Design of a constructed wetland to treat
without separation of biomass and medium. FEMS Microbiol Lett mine drainage at UKHM. http://technology.infomine.com/enviromine/
1992;92:181 – 6. wetlands/cw_cases.htm; 1995.
Saxena D, Gowri PM, Mago R, Srivastava S. Removal of copper by USEPA Mine Waste Technology Program. Activity III, Project 16: in-
Pseudomonas putida strain S4 isolated from copper mines. Indian J tegrated passive biological treatment process demonstration. Annual
Exp Biol 2001;39(6):590 – 3. Report. http://www.epa.gov/ORD/NRMRL/std/mtb/mwtp2001/
Scott JA, Karanjkar AM, Rowe DL. Biofilm covered granular activated index.html; 2001.
carbon for decontamination of streams containing heavy metals and USEPA Mine Waste Technology Program. Activity III, Project 12: sulfate-
organic chemicals. Miner Eng 1995;8:221 – 30. reducing bacteria reactive wall demonstration. Final Report. http://
Sen AM, Johnson DB. Acidophilic sulphate-reducing bacteria: candidates www.epa.gov/ORD/NRMRL/std/mtb/mtbdocs/actiiiproj12.pdf; 2002.
for bioremediation of acid mine drainage. In: Amils R, Ballester A, Valentine NB, Bolton HJ, Kingsley MT, Drake GR, Balkwill DL, Plymale
editors. Biohydrometallurgy and the environment toward the mining AE. Biosorption of cadmium, cobalt, nickel, and strontium by a Bacil-
of the 21st century. Process Metallurgy, vol. 9A. Amsterdam: Elsevier; lus simplex strain isolated from the vadose zone. J Ind Microbiol
1999. p. 709 – 18. 1996;16(3):189 – 96.
Senthilkumar S, Bharathi S, Nithyanandhi D, Subburam V. Biosorption of Valls M, Lorenzo VD. Exploiting the genetic and biochemical capacities of
toxic heavy metals from aqueous solutions. Bioresour Technol 2000;75: bacteria for the remediation of heavy metal pollution. FEMS Microbiol
163 – 5. Rev 2002;26:327 – 38.
Sharma S, Dastidar MG, Sreekrishnan TR. Zinc uptake by an isolated Veglio F, Beolchini F, Gasbarro A. Biosorption of toxic heavy metals: an
equilibrium study using free cells of Arthrobacter spp. Process Biochem Wong PK, Fung KY. Removal and recovery of nickel ion (Ni2 +) from
1997;32:99 – 105. aqueous solution by magnetite-immobilized cells of Enterobacter sp.
Volesky B. Advances in biosorption of metals: selection of biomass types. 4-2. Enzyme Microb Technol 1997;20(2):116 – 21.
FEMS Microbiol Rev 1994;14:291 – 302. Wong JPK, Wong YS, Tam NFY. Nickel biosorption by two Chlorella
Volesky B, Holan ZR. Biosorption of heavy metals. Biotechnol Prog species, C. vulgaris (a commercial species) and C. miniata (a local
1995;11:235 – 50. isolate). Bioresour Technol 2000;73:133 – 7.
Volesky B, May-Philips HA. Biosorption of heavy metals by Saccharomy- Yazgan A, Ozcengiz G. Subcellular distribution of accumulated heavy
ces cerevisiae. Appl Microbiol Biotechnol 1995;42:797 – 806. metals in Saccharomyces cerevisiae and Kluyveromyces marxianus.
Volesky B, May H, Holan ZR. Cadmium biosorption by Saccharomyces Biotechnol Lett 1994;16(8):871 – 4.
cerevisiae. Biotechnol Bioeng 1992;41:826 – 9. Yu Q, Kaewsarn P. Adsorption of Ni2 + from aqueous solutions by pre-
White C, Gadd GM. Uptake and cellular distribution of copper, cobalt and treated biomass of marine macroalga Durvillaea potatorum. Sep Sci
cadmium in strains of saccharomyces cerevisiae cultured on elevated Technol 2000;35:689 – 701.
concentrations of these metals. FEMS Microbiol Lett 1986;38(5): Yusef HH. Bioaccumulation of metal cations by free and immobilized cells
277 – 83. of Kluyveromyces marxianus. Adv Food Sci 1997;19(3/4):120 – 3.
White C, Sharman AK, Gadd GM. An integrated microbial process for the Zaluski MH, John MT, Canty MC, Baker MA. Field performance of en-
bioremediation of soil contaminated with toxic metals. Nat Biotechnol gineered SRB reactors for removing heavy metals. Proceedings of In-
1998;16(6):572 – 5. ternational Containment and Remediation Technology Conference and
Wilde EW, Benemann JR. Bioremoval of heavy metals by the use of Exhibition, Orlando, Florida; 2001 [http://www.containment.fsu.edu/cd/
microalgae. Biotechnol Adv 1993;11:781 – 812. content/srch_f_a.htm].

Metal Bioremediation Through Growing Cells PDF

Uploaded by

Copyright:

Available Formats

Metal Bioremediation Through Growing Cells PDF

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Metal Bioremediation Through Growing Cells PDF

Uploaded by

Copyright:

Available Formats

Environment International 30 (2004) 261 – 278

Metal bioremediation through growing cells

Received 1 May 2003; accepted 4 August 2003

Keywords: Metal; Growing cells; Bioremediation; Metal resistance

1. Introduction known quantities of pollutants in the water they consume.

3.1. Cadmium optimal strategy for metal remediation. In addition, several

A. Malik / Environment International 30 (2004) 261–278

You might also like