Indian Journal of Experimental Biology Vol. 41, September 2003, pp. 972-985
Bioremediation of chromium contaminated environments
Sara Parwin Banu Kamaludeen, K R Arunkumar, S A vudainayagam & K Ramasamy*
Fermentation Laboratory. Tamil Nadu Agricultural University, Coimbatore 641003, India
Bioremediation is the most promising and cost effective technology widely used nowadays to clean up both soils and wastewaters containing organic or inorganic contaminants. Discharge of chromium containing wastes has led to destruction of many agricultural lands and water bodies. Utilisation of chromium(Cr) reducing microbes and their products has enhanced the efficiency of the process of detoxification of Cr(VI) to CrUll). This review focuses mainly on the current technologies prevalent for remediation like natural attenuation, anaerobic packed bed bioreactors (using live cells, Cr(VI) reductases or their byproducts) and usc of engineered microorganisms. Treatment of wastewaters by biosorption or using biorilms and immobilized microbial cells are also discussed.
Keywords: Bioremediation, Chromium contamination, Chromium reducing microbes, Engineered microorganisms
Disposal of industrial and urban wastes to soil and water bodies has led to disastrous consequences to these ecosystems. Due to the excess loading of these wastes beyond their self cleaning capacities, these ecosystems has resulted in decreased availability of clean water to drink and normal soils for crop production. Enormous amount of organic and inorganic wastes from various industries has been disposed discriminately before the enactment of stringent regulations in many countries including India. Compared to the organic wastes, inorganic wastes, like heavy metals, pose a great threat, as they cannot be completely removed/degraded from the ecosystem like organic pesticides.
Chromium is an important heavy metal widely used in the metallurgic, refractory, chemical and tannery industries. Chrome plating, the deposition of metallic Cr, imparts a refractory nature to materials rendering them resistant to microbial attack and flexible over extended periods of timel. More than 170,000 tons of Cr wastes are discharged to the environment annually as a consequence of industrial and manufacturing activities2. Of the total Cr used in the processing of leather, 40% is retained in the sludge, disposal of which onto land and into water bodies has led to increased Cr levels reaching as high as 30,000 mg kg-J3
Detailed assessment of the tannery waste contaminated sites in Tamil Nadu, India and Mount Barker near Adelaide, Australia revealed the
"Corresponding author:
E-mail: ramasamytnau@yahoo.com, Fax: 0091-422-2431672
extensive contamination of soil and surface or ground water.4-7. Although no definite pattern was observed in different depths, it was reported that the concentration ranged between 100 mg to 70,000 mg kg-I in surface and subsurface soils of Tamil Nadu where old tanneries were located. The presence of high concentrations of soil chromium pose significant risks to animals and humans living in the vicinity of these sites through dust particles during summer rn Adelaide and winter months in India.
Distribution
Chromium is a potential soil, surface water, ground water, sediment and air contaminant. Soil chromium levels are usually related to the chromium level in the parent material, and except in soils derived from serpentine soil materials the natural background levels of soil chromium are actually lows.9. Chromium concentration in different environmental components is shown in Table 1.
High soil chromium levels are usually associated with anthropogenic contamination, mainly from industrial operations 10. Tanneries is one among the major industries and based on the processes used the composition of the sludge varies as well as the concentration of Cr (Table 2)11.
Speciation of chromium
Chromium toxicity and mobility depends on its oxidation state. Though chromium can exist in ox idation states ranging from 2- to 6+ 12, only chromium (VI) and chromium (111) are normally found within the range of pH and redox potentials
KAMALUDEEN el at.: BIOREMEDIATION OF CHROMIUM CONTAMINATION 973
Table I - Chromium concentration in different environmental components
Environmental component Continental crust Soil
Fresh water Sea water Drinking water Air samples
Concentration 80-200 mg/kg 10-150 mg/kg 0.1-6.0 mg/L 0.2-50.0 mg/L 0.05 mg/L 0.015-0.03 mg/m) Table 2 - Chemical composition of tannery chrome sludge from
different countries
Composition of tannery • South USA South
sludge Australia India
pH 7.71 * 7.8
EC (dS m-I) 3.5 * 3.5
Total organic carbon (%) II 15.4 9.6
Cr (%) 3.04 3.86 0.08
CaCO)(%) 11.7 * *
Ca (%) 4.7 1.8 0.8
Na (%) 4.7 0.7
*
Fe (mg kg-I) 1.1 * *
Mn (mg kg·l) 1.3
* *
Cu (mg kg-I) 66.5
*
*N (mg kg·l)
*
31000 4300P (mg kg-I) 4032
*
5100K (mg kg·l) 3549 1900 40
*Not reported
concern of various environmental systems (Fig. 1).
The trivalent forms are relatively immobile, more stable and much less toxic than hexavalent formsI3.14. Chromium, based on the physicochemical conditions, occur either as trivalent, Cr(l II) or hexavalent, Cr(VI) specIes.
Among this the hexavalent species, Cr(VJ), because of its mutagenic and carcinogenic nature, is considered as one of the priority pollutantsl5
. In the absence of reducing agents, Cr(VI) is soluble and hence mobile and it pose a great threat to surface and groundwater quality.
Transformation of chromium
Chromium is known to undergo various chemical and biological reactions in the natural systems that govern speciation of the metal and in turn its environmental behaviour. Important reactions include oxidationlreduction, precipitation/dissolution and absorption/ desorption. Both oxidation of Cr(llJ) and reduction of chromium(VI) can occur in geologic and aquatic environments.
ChemicaL transformatioll
Hexavalent chromium is a strong oxidation agent and is readily reduced in the presence of appropriate
electron donors. Rai and Zachara16
reported that Fe(II) actively reduced Cr(VI) and the reaction rate was dependent on the solubility of Fe compound. The presence of organic matter enhanced · Cr(VI) reductionI7.18. Low oxygen status also resulted in reduction of Cr(VI) and Cr(III)19.
Microbial transformation
The microbial inter conversions of heavy metals are of prime interest, since most of the heavy metals have entered the environment as a result of new industrial processes. The discovery of microorganisms that preferentially reduced hexavalent chromium has led to applications in the bioremediation of chromium contaminated ecosystems (Table 3). Bioreduction of chromate can occur under both aerobic2o
-22 and anaerobic conditions23,24.
Direct and indirect bioreduction and altered uptake appears to be a function of resistance to chromate.
Bioreduction of chromate OCCUlTed directly as a result of microbial metabolism or indirectly by the action of bacterial metabolites such as H2S20. Some of the important microbes involved in chromium reduction are Euglena gracilis, Pseudomonas aeruginosa, Enterobacter cloacae, Pseudomonas fluorescens, etc.
Anaerobic bacterial strains with accelerated Cr (VI) reducing capabilities have been isolated from chromate contaminated water and sludge23-26. As heavy metals occur naturally at high concentration in various environmental systems, resistance and tolerance mechanisms are being evolved within microbial communities. Certain Cr(VI) resistant strains like Enlerobacter sp and Pseudomonas sp.
reduced Cr (VI) to Cr (Ill) under anaerobic condi tions22.25.27.28.
Komori el al23 reported that Enlerobacter cloacae strain HOI reduced Cr(VI) anaerobically while growing on acetate, ethanol, malate, succinate and glycerol but its reduction efficiency was decreased significantly while the organism was grown on glucose, molybdate, vanadate and manganese oxide. It was further recorded that rate of reduction was proportionate to cell density, optimum pH and temperature were 7.0 -- 7.8 and 30-3rc.
Turick et al,z9 isolated hexavalent chromium reducing anaerobes from hexavalent chromium contaminated environments. These organisms were capable of reducing toxic and mobile Cr(VI) to less toxic and immobile Cr(lll). In addition the apparent ubiquity of Cr(VI) reducing bacteria in soils and sediments indicated the potential for III SilU
974 INDIAN J EXP BIOl, SEPTEMBER 2003
bioremediation of Cr(VI) contaminated soils and ground water. Defilippi and Lupton30 designed an anaerobic bioreactor utilizing marine derived, sulphate reducing bacteria, immobilized as a biofilm on gravel. Their experiments have suggested that with
Cr citrate
...
... ...
Cr (III) precipitales and polymers
citrate
additional clarification, Cr level can be brought down to as low as 0.01 mg L·t• This is due to the reduction of Cr(VI) by H2S produced by the sulphate respiring bacteria. The H2S produced by sulphate respiring bacteria in anaerobic systems diffused out into the
'"
HCrO; '"
'"
leaching plants uptake
'" )If adsorption/
'" '" precipitation
...
:' SUN ':
...
Fig. I- Chromium speciation in natural ecosystem"
Table 3 - Microbes used in bioremediation of Cr
Mechanism Organism
Bioreduction Pseudol7lonas ambigua G-l
Bioreduction Sulfate-reducing bacteria
Biorcduction Unidentified bacterial cultures
Bio!'cduction Chalamydomonas sp
Biosorplion Oscillmoria sp
Biosorplion ArlhrobaCler sp Agrobaclerium sp
Biosorption/Bioreduction Pseudomonas aeruginosa 5128
Biosorption/Bioreduction Sulfate-reducing bacteria
Source: modified from losi et al9
Description and Effectiveness
Cr(VI) concentration was lowered from ISO to 3S mg l·1 ovcr 36 hr in liquid media
Cr(VI) in water was lowered from II to <0.21 mg l·1 with addition of sulphate and acetate in an anaerobic bioreactor that produced H2 S, subsequently reducing the Cr(VI)
Reduced 40-60% of Cr (VI) added to liquid media at 10 and 25 mg l·1 approximately 50% at 60 mg l·1 and 10% at 120 mg l·1 over 18 hr with no additional nutrient supplement
An average of 30% Cr removed from water with initial concentrations of 0.2 mg L·1 in an aerobic bioreduction system Algal cultures removed 20% of Cr from water spiked at levels of 1-20 mg L·1
Accumulated Cr with increasing concentration gradient of Cr (VI) up 400 mg l·1 (Arthrobacter) and 100 mg l·1 (Agrobacterium )
Removed IS-SO% of Cr from liquid media spiked with 1,000 mg L·1 Cr (VI) over 82 hr. Addition of glucose enhanced the removal rate.
100% Cr removal was achieved in water with Cr (VI) up to ISOmgL·1
KAMALUDEEN el al.: BIOREMEDIATION OF CHROMIUM CONTAMINATION
975
medium and reduced Cr(VJ). The trivalent chromium thus formed react with OH· to form insoluble / immobile Cr(OH)3. This technique is also being evaluated as a possible in SilLi treatment for immobilization of Cr in chromate contaminated soils and ground water31.
Bioremediation
Bioremediation has been used as a strategy using microorganisms (introduced or indigenous) for complcte transformation of organic pesticides to harmless end products such as COl and H20. Likewise, microorganisms can transform inorganic pollutants, not necessarily completely, but to compounds with decreased solubility, mobility and toxicity. For instance, as stated in Table 3, microorganisms can transform toxic and reactive Cr(VI) to less toxic Cr(lll).
Cr(VI) bioremediation technology
A wide range of microorganisms exhibits an exceptional capacity to detoxify Cr(VJ) by converting it to less soluble and much less toxic Cr(lll). This capacity is harnessed in bioremediation technology for Cr(VJ) wherein the microbial strains are multiplied to desired population and pumped into soil/sediments in reactors to promote Cr reduction.
The bioremcdiation efficiency can be enhanced by supplements with organic matter and other nutrients in the water/soil to promote the growth of the introduced microorganisms. The addition of organic sources to the soil can promote the proliferation of indigenous Cr(Vl)-reducing microorganisms as well since Cr(VI) reducers, both aerobic and anaerobic, are ubiquitous in the soil environment. Losi el al.9 decontaminated largc volumes of Cr(VI)- contaminated water by passing it through an organic amended (cattle manure) soil. Indigenous soil microorganisms augmented by the organic amendments were largely involved in the reduction of Cr(VI) in the water, followed by precipitation and immobilisation of the Cr(lll) formed. In in SilLi
techniques. nutrients are pumped along with aeration to promote the Cr reduction by aerobic Cr(VI)- rcducing bacteria. Some Cr-reducing bacteria and algae have been efficiently used in the treatment of Cr-rich waste water9.32.3J B ioreactors are cost- effecti ve and are effective for decolltami nation of Cr(VI)-contaminated waste water. However, success has been limited for large scale decontamination of Cr(Vl)-pollulCd complex soils.
Recently, for treatment of soils enriched with chromite ore processing residue, a technique
involving the use of organic-rich acidic manure along with chrome reducing microbes to effectively reduce the Cr(VI) in the waste has been developed. This layer is positioned below the Cr-rich waste and Cr(Vl) leaching out of the waste, is effectively reduced in the organic layer, thereby preventing f urt h er contam1l1atlon . . 0 f groun d water· 3435 . .
As described by Losi el al.9, the bioremcdiation of the Cr(VI)-contaminated soil is achieved by either direct or indirect biological reduction. Most of the direct microbial reduction would be expected on surface soils. In the subsurface layers, indirect biological reduction of Cr(VI) involving H2S can be predominant and very effective, especially in situations where in situ stimulation of sulfate reducing bacteria is achieved through the addition of sulfate and nutrients. The H2S, diffused into inaccessible soil pores, promotes the reduction of not only Cr(VI), but also Mn oxides, involved in reoxidation of Cr(Ilf).
This method has shown some promise for remediation of Cr(VI) contaminated soils when applied to an anaerobic bioreactor system9.
Anaerobic packed-bed bioreactor
Anaerobic Cr(Vl)-reducing microorganisms are known to be ubiquitous in soils27. Anaerobic chromate reducing strains have been successfully used for the
d · d d· . f 29.36.37
re uctlon an se Imentatlon 0 tannery wastes . Turick and his group have developed an anaerobic bioprocess for Cr(Vl) reduction using a mixed culture of soil isolates or indigenous microorganisms in a packed-bed bioreactor containing ceramic packing or DuPont Bio-Sep beads3s.39
. There is evidence to suggest that organic contaminants such as aromatic compounds are suitable electron donors for Cr(Vl)
d . 40 Cl . d · . b h
re uctlon . lromlUIl1-re uClng micro es may t en be able to simultaneously remediate organic contaminants as well.
Scope for engineered microo"ganisms
Cr(VI) reduction by a wide range 01 microorganisms is of environmental and biotechno- logical significance. Bioremediation of chromate- polluted environments often poses two major problems: (i) inability of introduced Cr(V!)-reducing microorganisms to establish and function at sites polluted with mixtures of contaminants; and (ii) biodegradation rates not adequate enough to achieve acceptable residue levels within an acceptable time fralne. Several strategies have been proposed to enhance the rates of bioremediation of pollutants in
976 INDIAN J EXP BlOL, SEPTEMBER 2003
such inhospitable environments. One of the approaches is to develop engineered novel strains with increased Cr(VI)-reducing efficiency for such situations. Recently, Gonzalez41 cloned two bacterial genes encoding different soluble chromate reductases (Class I and Class II) that reduce Cr(VI) to Cr(lll). Each class has several close structural homologs in other bacteria. Five of these proteins, overproduced in pure form, ~ould reduce chromate and quinones. Class Il proteins could also reduce nitroaromatic compounds. Efforts are underway to use these genes and proteins directly in bioremediation of chromate polluted environments.
Natural attenuation
Natural attenuation involves in situ physical, chemical and biological processes to decrease the concentration of a contaminant in the environment over time without human intervention42.
Biotransformation plays a major role in the natural attenuation of several contaminants in long-term contaminated sites. In a long-term tannery waste contaminated site at the Mount Barker site in South Australia, industrial discharges of the waste ceased about 25 years ago. Analysis of samples revealed almost same Cr(VI) levels in the soil (around 40 mg kg·i) and water (up to 2 mg L·1) at 204 and 2543 years after the last waste input. Thus, during 5 years (1997- 2002), there was no appreciable natural attenuation of Cr(VI) at this site although the soil was rich in organic carbon (9.8-15.7%) and harbored Cr(VI) reducing microorganisms44. Incubation of this contaminated soil without and with added cow manure under saturated conditions led to complete disappearance of Cr(VI) within 20 days; but Cr(VI) reappeared probably due to reoxidation of Cr(TII) when the saturated soil was subsequently subjected to drying. However, no decrease in the concentration of Cr(VI) occurred in the Mount Barker soil held at 70%
water holding capacity even in the presence of cow manure. Although Cr(VI) can be reduced by a wide range of aerobic microorganisms (See Table I), its reduction in the contaminated soil occurred under saturated conditions and not at 70% water holding capacity. Evidently, reoxidation of Cr(IlI) and moisture stress conditions would probably explain the lack of natural attenuation of Cr(VI) In the contaminated soil at the Mount Barker site.
A wide variety of heterotrophic microorganisms is involved in the reduction of Cr(VI) to Cr(lll), aerobically or anaerobically depending on the
organism, both in soil and water environments~5. Cr(VI)-tolerant and sensitive bacteria, with ability to transform Cr(VI) to Cr(lll), occur widely in diverse ecological condition : water, sediments and soil31.
Evidence suggests that Cr(VI) reducing microorganisms are ubiquitous in soils and can enhance the detoxification of Cr(VI) under ideal physico-chemical conditions29.
Direct Cr(VI) reduction
In soils, microbial Cr reduction may occur directly or indirectly. In the direct mode, Cr is taken up by the microbes and then enzymatically reduced9.23
.46, while in the indirect mode, products (reduction or oxidation) of microbial decomposition in the soil such as H2S mediate the reduction of Cr(VI)30. Direct microbial reduction of Cr(VI) was first reported in 1970s24,47 when certain Pseudomonas strains, isolated from chromate-containing sewage sludges, could reduce chromate, dichromate and crocoite during anaerobic growth. Since then, several bacteria with exceptional ability to reduce Cr(VI) have been isolated from Cr- contaminated and uncontaminated soil samples.
Microorganisms, implicated in direct or indirect reduction of Cr(VI), are listed in Table 1.
Cr(VI) reduction in microbial cultures
Since the first reports of isolation of facultative anaerobic Cr(VI)-reducing bacteria in mid-1970s24,
literature is abundant with instances of the reduction of Cr(VI) by several microorganisms, bacteria in particular48, mostly isolated from Cr-impacted environments (Table I). Strains of Oscillatoria, Chlorella and Zoog/oea have also been reported to enzymatically reduce Cr(VI)9. But, as noticed with bacterial resistance to Cr(VI), Cr(VI)-reducing bacteria have been isolated also from environments with minimum or no impact of Cr29.49.50. It is also interesting to note that pure cultures of microorganisms, not previously exposed to Cr(VI), were capable of reducing it49. Although the exact mechanism is not known, microorganisms capable of reducing Cr(VI) aquired the enzymes for degrading related compounds present in the environment or produce the reductants that in turn reduce Cr(Vl) by chemical redox reactions. Anaerobic chromate reducing strains are prevalent in subsurface soils and probably enhance Cr reduction in this environment2Y•
Cr(VI)-resistance and reduction are not necessarily interlinked. Cr(VI) may be reduced by both Cr(VI)- resistant and Cr(V l)-sensi ti ve strains of bacteria and
KAMALUDEEN el al.: BIOREMEDIATION OF CHROMIUM CONTAMINATION 977
not necessarily all Cr-resistant bacteria can reduce Cr(VI). For instance, some aerobic Cr(VI)-resistant bacteria were not capable of reducing itso.sl. Cr(VI) reduction in aerobic conditions may not be a resistance mechanism in bacteria, but a "trivial side activity of the reductase that may have evolved on other substrates27. In a bioprocess strategy for effective bioremediation of Cr(VI), it is important to use Cr(VI)-resistant microbes, with ability to reduce it. Two strains of Pseudomonas jluorescens, one resistant and the other sensitive to Cr(VI), reduced Cr(VI) at comparable rates20.48. Likewise, three Cr(VI)-sensitive bacteria from an uncontaminated soil and three Cr(VI)-resistant bacteria from two metal- stressed foundry soils and a tannery readily reduced Cr(VI) anaerobicall/ 9. Interestingly, Cr(VI)-sensitive BaciLLus sp. from the uncontaminated soil was the most effective in reducing Cr(VI) among the three Cr(VI)-resistant bacterial strains from metal stressed soils and three Cr(VI)-sensitive bacterial strains from the uncontaminated soil. These bacteria grew aerobically in acetate minimal medium supplemented with sodium chromate, but reduced Cr(Vl) only anaerobically. in the suspension of resting cells of aerobically grown bacteria. Anaerobic growth of the bacterium at the expense of Cr(VI) as electron acceptor was negligible. Conversely, an Arthrobacter sp., isolated from a long-term tannery waste contaminated soil, was resistant to Cr(VI) at 100 tLg/mL, but could not reduce it at this concentration44.
Likewise, Cr(VI) reduction occurred equally rapidly with both Cr(VI)-resistant and plasmid-cured Cr(VI)- sensitive strains of P. jluorescenio. Chromate resistance determinants have been described on plasmids in several bacteria, especially in Pseudomonas. But, Cr(VI) reduction determinants have not been found on plasmids. Cr(VI) reduction was independent of chromate resistance, conferred by plasmid pLHB 1, in P. jlourescenio. But, in P.
mendocinas2, plasmid pARI 180 determined both chromate resistance and Cr(VI) reductions3.
Microorganisms, known to reduce Cr(VI), reduce it under aerobic and I or anaerobic conditions, but the physiological role in such transformations is not clear.
Earlier reports24.26 have shown that facultative anaerobes (Pseudomonas and Aeromonas strains) reduce Cr(Vl) to Cr(lII) anaerobically. Anaerobic bacteria with great Cr(VI)-reducing potential are ubiquitous in both Cr(VI)-contaminated and uncontaminated soils29
.54. There is no convincing evidence yet to suggest that Cr(VI) serves as the
electron acceptor to support the anaerobic growth of bacteria. E. cloacae grew well under aerobic conditions and slowly under anaerobic conditions at chromate concentrations above 10 mM in nutrient broth, but could reduce chromate only under anaerobic conditionsso. Also, there is evidence that O2
inhibited the reduction of Cr(VI) by Enterobacter cloacae strain HO I in a medium containing other carbon sources as electron donors23.5o. Likewise, E.
coli could reduce Cr(VI) only in the absence of 02SS.S6. Under anaerobic conditions, Cr(VI) serves as a terminal electron acceptor through electron transport systems involving cytochrome c in Enterobacter cloacaeso, cytochrome band d in Escherichia colis7 and cytochrome c3 in Desulfovibrio vulgaris45.
Membrane or soluble fractions may be involved in the reduction of Cr(VI). Under aerobic conditions, both NADH and endogenous cell reserves may serve as elecron donors for Cr(VI) reduction. A recent stud/8 established a relationship between the bioavailability of H2 and chromate reduction in anaerobic aquifer sediments. The anaerobic enrichment, developed from the sediment, utilized Cr(VI) and was dependent on H2 for growth and chromate reduction. In the absence of Cr(VI), H2 accumulated in the anaerobic medium. But, under Cr(VI) reducing conditions, no H2 and methane accumulated due to utilisation of the H2 by the enrichment. When H2 was provided in the medium as the electron donor the enrichment could reduce 40 mg L-1 Cr(VI) in 6 days. Increasing the availability of H2 by addition of suitable electron donors (formate, H2 and glucose) accelerated the reduction of chromate.
Gram positive bacteria, capable of reducing Cr(Vl) as a terminal electron acceptor and with a relatively high level of resistance to chromate, have been isolated from tannery effluents II.S9.60. A chromate- resistant Gram positive bacterium (ATCC 700729) tolerated high concentrations (up to 80 mg/mL) of dichromate and reduced 87% of the Cr(VI) in 20 mg K2Cr207/mL in 72 hr in a nutrient-rich mediumS'>. The bacterium could reduce Cr(Vl) even at a concentration of dichromate as high as 80 mg/mL, but took longer time for its reduction at 80 mg/mL than that required at 20 mg/mL. Chromate reduction occurs either anaerobicall/0.49.5o.61, aerobically17.61, and. under both conditions62
. Agrobacterium radiobacter EPS-91663 and Escherichia coli A TCC 33456 could reduce Cr(VI) under both aerobic and anaerobic conditions. Likewise, a pseudomonad, isolated from a wood preservation site contaminated
978
INDIAN J EXP BIOL, SEPTEMBER 2003with chromated copper arsenate, reduced chromate under both aerobic and anaerobic conditions62. Also, Cr(YJ)-tolerant (>400 jkg/mL) facultative anaerobes (five isolates of Aerococcus sp.; two isolates of Micrococcus sp.; and one isolate of Aeromonas sp.), isolated from tannery effluent, apparently reduced Cr(YI) both anaerobically and aerobicall/,4. Cr(YI) reduction by these facultative anaerobes in diluted peptone water was more pronounced under anaerobic conditions (73-94% reduction) than under aerobic conditions (18-63% reduction). But conditions used for anaerobic and aerobic systems have not been described. Since peptone alone may catalyse chemical reduction of Cr(YI)6! it was not clear whether the Cr(YI) reduction in mjcrobial cultures was caused chemically, microbially or both. Cell suspensions of Pseudomonas pUlida PRS 2000, P. jluorescens LB303 and Escherichia coli AC80 aerobically reduced Cr(YI) to Cr(1II)27. Reduction of Cr(YI) in cell suspensions of these bacteria was more rapid and complete aerobically than anaerobically. After disruption of the cells of P. pUlida and centrifugation, the supernatant, but f10t the membrane fraction (pellet), reduced all the added Cr(Yl) within 1 hr.
Likewise, Wang and Shen6! reported that resting cells of Bacillus sp. and Pseudomonas fluorescens LB300 aerobically reduced Cr(YJ). But, Cr(YI) reduction by the cells of Escherichia coli was inhibited in the presence of oxygen55.56
. Enlerobacler cloacae, a chromate resistant strain could grow in the presence of Cr(YI) under both aerobic and anaerobic conditions, but Cr(YI) was reduced only anaerobicall/o. The strain lost both resistance and Cr(YI)-reducing ability on anaerobic growth on nitrate.
Cifuentes el al.32 reported that organic amendments enhanced the reduction of Cr in soils by indigenous microflora. Generally, Cr(YI) reduction by growing bacterial cells has been demonstrated in media containing natural aliphatic compounds, amino acids and fatty acids as electron donors6!. Microbial reduction of Cr(YI) occurred during anaerobic degradation of benzoate4o. Dissimilatory metal- reducing bacterium, Shewanella olleidensis could reduce Cr(Vl) when grown on fumarate or nitrate as an electron acceptor and lactate as an electron dono/'s. Cr(YI) reduction under fumarate and denitri fyi ng conditions, dependent on the physiological state of the culture, ,vas possibly inducible under anaerobic conditions. Cr(VI) reduction in the anaerobically grown stationary phase
of this bacterium is a complex process, possibly involving more than one pathwa/ 6.
A wide range of organic pollutants such as phenol, 2-chlorophenol, p-cresol, 2,6-dimethylphenol, 3,5- dimethylphenol, 3,4-dimethylphenol, benzene and toluene can also serve as electron donors for Cr(VI) reduction in cocultures contallllllg E. coli A TCC33456 and P. pUlida DMP_14o. Metabolites produced during phenol degradation by P. pulida served as electron donors for Cr(VI) reduction by E.
coli. Technology using such cocultures would help to simultaneously detoxify both organic pollutants and the toxic Cr(VI).
Non-metabolising resting cells of bacteria could reduce Cr(VI), but only in the presence of an added carbon source20.56,67. Killed resting cells could not cause Cr(VI) reduction56.6
!. Soluble enzymes in cell extracts can reduce Cr(VI) in the presence22. 67 or absence2o. 56 of added electron donors.
According to very recent evidence, nonmetabolic Cr(VI) reduction can occur on bacterial surfaces even in the absence of externally added electron donors in the meoium. Thus, Fein el al.68 demonstrated that non metabolizing cells of Bacillus subtilis, Sporosarcina ureae and Shewanella putrefaciens could reduce significant amounts of Cr(VI) in the absence of externally supplied electron donors. The Cr(VI) reduction by the bacterial strains was dependent on solution pH, decreasing with increasing pH, and presumably occurred at the cell wall and independent of the oxidation of bacterial organic exudates. Such non metabolizing reduction of Cr(VI) by bacteria in nutrient-poor conditions may be important in the biogeochemical distribution Cr.
Cr(VI) reduction by microorganisms, known to occur under both aerobic and anaerobic conditions (see Table 1), is a redox-sensitive process56.69
. The ability of washed resting cells of Agrobacterium radiobacter EPS-916 to reduce Cr(VI) was governed by their redox potenial63
. Resting cells of A.
radiobacter EPS-916, pregrown under aerobic conditions on glucose, fructose, maltose, lactose, mannitol or glycerol as the sole carbon and energy source, exhibited similar redox potentials of around - 200 mV and completely reduced 0.5 mM chromate.
On the other hand, the inability of the resting cells of the bacterium, pregrovm on glutamate or sllccinate, to reduce chromate was associated with relatively high redox potentials of -138 to -132 m V. Moreover, resting cells, pregrown under anaerobic conditions on glucose, had lower redox potentials (-240 mY) and a
KAMALUDEEN et at.: BIOREMEDIATION OF CHROMIUM CONTAMINATION 979
more pronounced chromate-reducing activity than did the aerobically grown resting cells on glucose with redox potential of -200 mY. Likewise, cells, pregrown anaerobically on chromate as the electron acceptor, effected more rapid reduction of chromate than did the anaeorobically grown cells ( -198 mY) on nitrate.
Evidence suggested a negative correlation between chromate reduction by the resting cells of A.
radiobacter EPS-9\6 and their redox potential. But, on the other hand, in an anaerobic enrichment from aquifer sediment, Cr(YI) reduction appears to occur under nitrate reducing conditions, but before iron and sulfate reduction58. Evidently, highly reducing conditions, necessary for the reduction of iron and sulfate and methanogenesis, may not be required for chromate reduction. However, it was observed that there existed a competitIOn among chromium reduction and nitrogen reduction. Clostridium and Methanosarcina reduced both nitrogen and chromium. In the presence of chromium nitrogen reduction was reduced 70. Actual mechanism of competition needs verification.
Abiotic reduction of Cr(YJ) has also been demonstrated in media rich in nutrients containing some reductants, especially under predominantly reduced conditions. Thus, even in sterile tryptic soy broth, Cr(Yl) was reduced abiotically with time as a function of redox potential29
. Thus, more than 50% of the 25 Ilg Cr(YI)/mL added to the tryptic sterile broth was reduced abiotically in 60-80 hr at redox potentials of -120 and -380 mY, as compared to <27% reduction at +243, +186 and +58 mY during the corresponding period. It is therefore necessary to have appropriate control to exclude the chemical redox reactions when nutrient-rich growth media are used to assess the Cr(VI)-reducing ability of pure cultures of
. .
microorgalllsms.
Cr(VI) reductases
Cr(YI) reduction is mediated enzymatically (direct) and/or non-enzymatically (indirect). There IS
considerable literature on the invoivement of Cr(YI) reductases in direct reduction of Cr(Yl) to Cr(IIJ) by bacteria. In growing cultures with added carbon sources as electron donors and in cell suspensions, Cr(YI) reduction can be predominantly aerobic or anaerobic, but general I y not both. I nteresti r.gl y, Cr(YI) reductases can catalyse reduction of Cr(YI) to Cr(lII) anaerobicall/ 5, aerobically27.71 and also both anaerobically and aerobicall/ o.57.62.72. The Cr(YI)- reductase enzyme may be present in the membrane
fraction of the cells as in Pseudomonas fluorescens and Enterobacter cloacae28 or in the soluble fraction of the cells (cell-free system) as in P. ambigua22, P.
putida27 and a Bacillus Sp.72, with NADH, NADPH or H2 (Desulfovibrio vulgaris) as electron donors and possible involvement of cytochromes b, c and d.
Membrane vesicles of E. cloacae, reduced with NADH and then exposed to Cr(YI), oxidized c and b cytochromes and reduced Cr(YI). Evidence suggested that specifically cytochrome c548 was involved in the reduction of Cr(YI) by membrane vesicles73. In the presence of H2 and excess of hydrogenase, cytochrome c3, a periplasmic protein, in the soluble cell-free fraction of the cells in D. vulgaris45 reduced Cr(YI), 50 times faster than did the Cr(Yl) reducta~e
of P. ambigua with NADH and NADPH as electron donor22. Soluble fractions of the cell-free extract, largely cytoplasmic, of a pseudomonad from a wood preservation site reduced chromate, added at 10 mg Cr(YI)L-\, under both aerobic (55%) and anaerobic (80%) conditions in 2.5 hr62. Cr(YJ) reductase in anaerobically grown Shewanella putrefaciens MR-l was formate-dependent with highest activity in cytoplasmjc membrane74. The Cr(YI) reductase in P.
ambigua71 and a Bacillus Sp.72 have been purified and characterised. More recently, to clone a chromate reductase gene, a novel soluble chromate reductase of P. putida has been first purified to homogeneity and characterized, using ammonium sulfate precipitation, anion-exchange chromatography, chromatofocusing and gel filtration75
. The reductase activity was NADH- or NADPH-dependent. The optimum conditions for the chromate reductase were: 80°C and pH 5.0. Kinetic properties of the enzyme showed Km of 374 IlM CrO/ -and Ymax of 1.72 Ilmollmin/g of protein. Suzuki el al.71 sequenced the gene encoding the chromate reductase71 from P. ambigua. But, the genes encoding the chromate reductase in P. ambigua and P. pUlida were not homologous. A bacterial reduction of chromate by a flavin reductase with flavin is known 76. Furthermore the end product was a soluble stable Cr(lII)-NAD complex instead of Cr(lIl) precipitate. Since intracellularly formed CrOll) forms adduct product with DNA, protein, glutathione, and ascorbate in eukaryotic cells, the reported bacterial flavin dependent reductase in bacteria will protect the cells
Reduction products
Generally, in bacterial cultures or in enzyme systems, Cr(YI) is reduced to Cr(lll) without
980 INDIAN J EXP BIOL, SEPTEMBER 2003
transitory accumulation of any intermediate. But, there are instances when Cr(V) accumulates as a transitory intermediate during microbial conversion of Cr(VI) to Cr(IlI). For instance, the NADPH- dependent Cr(VI) reductase in P. ambigua catalysed the transitory formation of Cr(V) during conversion of Cr(VI) to Cr(1lI)71. Toxicity of Cr(VI) to microorganisms is probably associated with the transient formation of Cr(V) as an intermediate. Cr(V) is formed also during reduction of Cr(VI) in algal cultures and in reactions with physiological reducing agents such as NADPH, glutathione and several pentoses 77.
In most studies, conclusions on microbial reduction of Cr(VI) were based on its disappearance ancl/or accumulation of the Cr(IlI) [determined as the difference in total Cr and Cr(VI)] as the reduction product with incubation. The colorimetric diphenylcarbazide method commonly used in Cr(VI) estimation is not specific since its probable reduction product Cr(V) and hexavalent forms of Mo, V and Hg can also react with the same reagent. But, the direct measurement of the oxidation state of the Cr during bacterial reduction of Cr(VI) has not been attempted until recently. Daulton el al.78 used the electron energy loss spectroscopy (EELS) technique to characterize the oxidation state of Cr during Cr(VI) reduction by Shewanella oneidensis in anaerobic cultures. TEM of the cells exposed to Cr(VI) showed that the cells were encrusted in Cr-rich precipitates, mostly restricted to the outer surface of the cells.
These precipitates, based on analysis by EELS, contained Cr(Ill) or its lower state of oxidation.
Myers el al.74, using electron paramagnetic resonance (EPR) spectroscopy, confirmed the formation of Cr( V) via one-electron reduction of Cr(VJ) as the first step by a facultative anaerobe Shewanella pUlrefaciens MR-I.
Indirect reduction
Apart from the direct (enzymatic) reduction of Cr(VI), microorganisms can also mediate the reduction of Cr(VI) indirectly, involving a biotic- abiotic coupling. For instance, Fe(ll) and S2., produced by microorganisms through dissimilatory reduction pathways, can chemically catalyse several biogeochemical processes including Cr(VI) reduction 79.80. Fe(IIJ), an important electron acceptor for microbial oxidation of organic compounds (aliphatic and aromatic), is one of the most abundant metals in the soil. A wide range of bacteria couple the
oxidation of organic compounds and H2 to reduction of Fe(III) and S04 to Fe(II) and H2S, respectively under oxygen-stress conditions8o. This occurs as in submerged rice soils for example. Different genera of Fe(III)-reducing bacteria reduce Fe(JU) via different mechanisms81
. Recently, Wielinga et al.82 demon- strated the reduction of Cr(VI) by a biotic-abiotic coupling mechanism involving iron reduction.
Dissi mi latory Fe(Ill) reduction by Shewanella alga ATCC 51181, a facultative anaerobic bacterium, under iron reducing conditions provided a primary pathway for chemjcal reduction of Cr(VI), injected into a bioreactor, by microbially induced ferrous ion.
However, it has been difficult to differentiate the exact contribution between biological (direct) and chemical (indirect: biotic-abiotic) reduction of Cr(VI) in a soil environment. Evidence using Desulfovibrio vulgaris· as a model chromate reducer suggests that chemical reduction of chromate by Fe(II) was 100 times faster than that by D. vulgaris, a chromate reducer82
. In anaerobic environments abundant in Fe(Il), nonenzymatic reduction of Cr(VI) by Fe(lI) can be as important as enzymatic Cr(VI) reduction83. A facultative anaerobe Pantoea agglomerans SP L
coupled anaerobic growth on acetate and other electron donors to the dissimilatory reduction of electron acceptors, Fe(III), Mn(IV) and Cr(VI), but not sulfate84
. When Cr(VI) was added to this y- protobacterium culture with elemental sulfur alone, SO-disproportionation to sulfate and hydrogen sulfide occurred with concomitant growth of the bacterium and reduction of Cr(VI)85. Likewise, P. agglomerans SP 1 grew chemolithoautotrophically by the SO- disproportionation, coupled to reduction of Fe(III) and Mn(lV). Probably, SO-disproportionation that may be widespread in certain anaerobic environments may provide an effective mechanism for attenuation of Cr(VI) through its reductive detoxification.
Sui fate-reducing bacteria (obligate anaerobic heterotrophs) couple the oxidation of organic sources to the reduction of sulfate to sulfide. Reduction of Cr(VJ) by bacterially produced hydrogen sulfide, followed by precipitation of the Cr(lll) formed, is an important mechanism in sulfate-rich soil environ-
h b· d" '1318687'
ments w en an aero IC con Itlons preval . . , as In
flooded compacted soils. Likewise, sulfide, produced by sulfate-reducing bacteria, has been implicated in Cr(VI) reduction in marine environments88. Hydrogen sulfide, produced in acid sulfate soil under reducing conditions, is easily precipitated as FeS in reduced soils89 and sediments. Fe(II)90 and H2S87, both
+
KAMALUDEEN et 01.: BIOREMEDIATION OF CHROMIUM CONTAMINATION 981
microbially produced, are effective reductants of Cr(VI) under reduced conditions as is the FeS91. Low concentrations of Cr(Vl) can accelerate the growth and sulfate-reducing activity of sulfate-reducing bacteria92 and thereby the reduction Cr(VI) by the H2S evolved. Interestingly, a spore-forming sulfate- reducing bacterium, Desulfotomaculum reducens sp.
nov. strain MI-l, isolated from sediments with high concentrations of Cr and other heavy metals by enrichment, could grow with Cr(Vl) as sole electron acceptor in the absence of sulfate with butyrate, lactate or valerate as the electron donor9J. Cr(VI) was presumably reduced to Cr(lII) as Cr(OHh In the absence of Cr(VI), no bacterial growth was noticed.
Biologically generated sulfur compounds with high reducing power such as sulfite, thiosulfate and polythionate can catalyse the chemical reduction of Cr(VI). Chemoautotrophic ba~teria, belonging to Thiobacilli group, that can derive energy from the oxidation of inorganic sulfur compounds during sulfur oxidation, generate a range of sulfur compounds such as sulfite and thiosulfate with high reducing power that can in turn catalyze the reduction of Cr(VI). For instance, Thiobacillus ferroxidans, grown on elemental sulfur, has been used to reduce Cr(VI) under aerobic conditions94.95. The Cr(VJ)-reducing ability of the cells of this bacterium under aerobic conditions in shake flasks and in fermentation vessels was related to the generation of protons with high reducing power from elemental sulfur96. T.
ferroxidans could reduce Cr(VI) over a wide pH range (2-8), interestingly with more pronounced reduction at lower pH, associated with increased oxidation of elemental sulfur to products with high reducing power. Cr(VI) reduction, mediated by T.
ferroxidans in the presence of elemental sulfur, occurred under both aerobic and anaerobic conditions, but more effectively under aerobic conditions.
Evidently, bacterial reduction of Cr(VI), involving biotic-abiotic coupling, can occur under both sulfate- reducing and sulfur-oxidising conditions. Thus, Cr(VI) reduction or immobilisation can be effected abiotically by different substances; but, there is considerable progress in recent years on the feasibility of using biological reduction for treatment of Cr(VI)- containing wastes.
Several remediation techniques are available for soils contaminated with Cr. Technology applicable to a particular Cr site depend on the clean up goals, the form of Cr present and volume and physico-chemical conditions of the contaminated environments like soil,
water and sediments. Processes, developed for remediation of environments contaminated with chrome wastes, are more suited for aquatic systems than for terrestrial systems. Traditional methods, used especially for waste waters, involve chemical or electrochemical reduction of Cr(VI) to Cr(lll), precipitation of the latter and its removal by filtration or sedimentation9o. Chemical methods are generally not cost effective and may themselves generate hazardous byproducts79. Microorganisms are capable of altering the redox state of Cr by reducing Cr(VI) to Cr(lll) through direct (enzymatic) or indirect (via iron reduction, sulfate / sulfur reduction or sulfur oxidation) processes.
Bioremediation technologies for waste water/solution
Biosorption
Sequestration and immobilisation of heavy metals, especially in the solutions of effluents and waste water, can be accomplished through biosorption, a passive process of metal uptake, using biomass (dead biomass in particular)97. Biosorption is essentially a non-directed physico-chemical complexation reaction between dissolved metal species and charged cellular components, that involves sorption and/or complexing of metals to living or dead cells. The precipitation or crystallisation of metals leading to their sequestration can take place at or near the cell. Also, insoluble metal species can be physically entrapped in the microbially produced extracellular matrix or precipitated 111 bacterial or algal exudates98
. Extracellular matrices may consist of neutral polysaccharides, uronic acids, hexosamines and organically bound phosphates that are capable of complexing metal ions. Metabolically mediated accumulation is usually intracellular and linked to the control of plasmid linked genes99.
Yeasts and bacteria as well as algae can effectively sequester metals in solutions1OO, because of their metal-binding capabilities. Algae, such as Scenedesmus, Selenastrum and Chiarella, are known to bioaccumulate metals101. The functional groups present in the cells and cell walls of fungi and algae can serve as the probable sites for biosorption of metals. For instance, the amino group of chitin (R2- NH) in alga Sargassam and chitosan (R-NH2) in fungi are probably the effective binding site for Cr(VI). But, functional groups such as chitin and chitosan seem to contribute only 10% of the metals sequestered by the biomass.
982 INDIAN J EXP BIOL, SEPTEMBER 2003
Biosorption, using especially dead biomass, is a cost-effective technology for removal of heavy metals, and is as effective as ion exchange, but is yet to be exploited commercially. Biosorption research was confined to mostly cations and there is a need for research on uptake of anions by biomass such as Cr.
Biosorption of Cr(VI) is often followed by its bioreduction to less toxic Cr(III) and eventual precipitation of the latter. Bioreduction has been used for removal of Cr(VI) from wastewater systems in METEX anaerobic sludge Oreactor, BIO-SUBSTRAT anaerobic micro-carrier reactor and Agarkar Research Institute chromate reduction process 102. Biosorption is not suitable for detoxification of solid Cr-wastes in soils.
Biofllms in bioreactors
Bacterial biofilms have been recommended as an efficient means of remediating contaminants in the environment, because, biofilms provide tolerance to desiccation, a high level of pollutants and other stress factors. Smith and Gadd,03 used a mixed culture sulfate reducing bacterial film for reduction of hexavalent Cr. In the presence of lactate as the carbon source and sulfate, 88% of the 500 /lmol of Cr(VI) was removed from the solution with bacterial biofilm as insoluble Cr(lll) in 6 hr. Since sulfide, a reductant of Cr(VI), was not detected in the medium and no reduction occurred in uninoculated medium, dissimilatory chemical reduction was not involved in Cr(VI) reduction. Evidently, Cr(VI) reduction in sulfate reducing bacterial films was biologically mediated, presumably by enzymes. It is also possible to recover the insoluble or precipitated Cr(I1l) from the bacterial films. There is scope for using this biofilm technology for detoxification of Cr wastes in a bioreactor.
lmmobilised cells
Cells immobilised on polyacrylamide gel can be used for effective detoxification and removal of metals in solution from effluents in a reactor. Intact cells of a sulfate reducing bacterium Desulfovibrio desuljilricans, immobilised on polyacrylamide gel, reduced around 80% of 0.5 M Cr(VI) with lactate or H2 as the electron donor and Cr(Vl) as the electron acceptorl04. Insoluble Cr(IlI) accumulated on the surface or interior of the gel. Immobilised cells also effected the reduction of other oxidised metals, Mo(VI), Se(VI) and U(V!). lmmobilised cells may be useful for detoxification of Cr(VI) in bioreactors.
Bioreactor using living microorganisms
Rajwade and Paknikar,05 developed an efficient chromate reduction process using a strain of Pseudomonas mendocina MCM B-180 for treatment of chromate-containing wastewater. The bacterial strain used was resistant to 1600 mg Cr(VI)L" and reduced 2 rnM chromate [100 mg Cr(Vl) L·I] in 24 hr.
In 20-mL continuously stirred bioreactors containing this bacterium and sugarcane molasses as a nutrient, 25-100 mg chromate L'I was removed within 8
hr52"o2. Efficiency of this bioremediation process is
enhanced by anaerobiosis.
Conclusion
Compared to other conventional methods, bioremediation is highly economical and ecofriendly as this generates no further waste into the environment. Challenges till remain in the form of elevated concentrations of Cr(VI) in groundwater and in deeper soil profiles that needs further research in the field of bioremediation.
References
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2 Gadd G M & White C. Microbial treatment of metal pollution - A working biotechnology? Trends Biorechnol, II (1993) 353.
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