Biosorptive removal of cadmium from contaminated groundwater and industrial effluents

The cadmium removing capacity of a biosorbent Calotropis procera, a perennial wild plant, is reported here. The biomass was found to possess high uptake capacity of Cd(II). Adsorption was pH dependent and the maximum removal was obtained at two different pH i.e. pH 5.0 and 8.0. Maximum biosorption capacity in batch and column mode was found to be 40 and 50.5 mg/g. The adsorption equilibrium (> or =90% removal) was attained within 5 min irrespective of the cadmium ion concentration. Interfering ions viz. Zn(II), As(III), Fe(II), Ni(II) interfered only when their concentration was higher than the equimolar ratio. The Freundlich isotherm best explained the adsorption, yet the monolayer adsorption was also noted at lower concentrations of Cd(II). The FTIR analysis indicates the involvement of hydroxyl (-OH), alkanes (-CH), nitrite (-NO(2)), and carboxyl group (-COO) chelates in metal binding. The complete desorption of the cadmium was achieved by 0.1M H(2)SO(4) and 0.1M HCl. The C. procera based Cd(II) removal technology appears feasible.     

Isolation and characterization of Staphylococcus sp. strain NBRIEAG-8 from arsenic contaminated site of West Bengal

Arsenic contaminated rhizospheric soils of West Bengal, India were sampled for arsenic resistant bacteria that could transform different arsenic forms. Staphylococcus sp. NBRIEAG-8 was identified by16S rDNA ribotyping, which was capable of growing at 30,000?mg?l(-1) arsenate [As(V)] and 1,500?mg?l(-1) arsenite [As(III)]. This bacterial strain was also characterized for arsenical resistance (ars) genes which may be associated with the high-level resistance in the ecosystems of As-contaminated areas. A comparative proteome analysis was conducted with this strain treated with 1,000?mg?l(-1) As(V) to identify changes in their protein expression profiles. A 2D gel analysis showed a significant difference in the proteome of arsenic treated and untreated bacterial culture. The change in pH of cultivating growth medium, bacterial growth pattern (kinetics), and uptake of arsenic were also evaluated. After 72?h of incubation, the strain was capable of removing arsenic from the culture medium amended with arsenate and arsenite [12% from As(V) and 9% from As(III)]. The rate of biovolatilization of As(V) was 23% while As(III) was 26%, which was determined indirectly by estimating the sum of arsenic content in bacterial biomass and medium. This study demonstrates that the isolated strain, Staphylococcus sp., is capable for uptake and volatilization of arsenic by expressing ars genes and 8 new upregulated proteins which may have played an important role in reducing arsenic toxicity in bacterial cells and can be used in arsenic bioremediation.     

Enhanced Adsorption of Trivalent Arsenic from Water by Functionalized Diatom Silica Shells

The potential of porous diatom silica shells as a naturally abundant low-cost sorbent for the removal of arsenic in aqueous solutions was investigated in a batch study. The objective of this work was to chemically modify the silica shells of a diatom Melosira sp. with bifunctional (thiol and amino) groups to effectively remove arsenic in its toxic As(III) form (arsenite) predominant in the aquatic environment. Sorption experiments with this novel sorbent were conducted under varying conditions of pH, time, dosage, and As(III) concentration. A maximum adsorption capacity of 10.99 mg g^-1 was achieved within 26 h for a solution containing 12 mg L^-1 As(III) at pH 4 and sorbent dosage of 2 g L^-1. The functionalized diatom silica shells had a surface morphological change which was accompanied by increased pore size at the expense of reduced specific surface area and total pore volume. As(III) adsorption was best fitted with the Langmuir-Freundlich model, and the adsorption kinetic data using pore surface diffusion model showed that both the external (film) and internal (intraparticle) diffusion can be rate-determining for As(III) adsorption. Fourier transform infrared spectroscopy (FTIR) indicated that the thiol and amino groups potentially responsible for As(III) adsorption were grafted on the surface of diatom silica shells. X-ray photoelectron spectroscopy (XPS) further verified that this unique sorbent proceeded via a chemisorption mechanism through the exchange between oxygen-containing groups of neutral As(III) and thiol groups, and through the surface complexation between As(III) and protonated nitrogen and hydroxyl groups. Results indicate that this functionalized bioadsorbent with a high As(III) adsorption capacity holds promise for the treatment of As(III) containing wastewater.     

Characteristics of molybdate-impregnated chitosan beads (MICB) in terms of arsenic removal from water and the application of a MICB-packed column to remove arsenic from wastewater

The removal of arsenic (As) species, such as As(III) and As(V), from water by molybdate-impregnated chitosan beads (MICB) in both batch and continuous operations was studied. The effects of pH, temperature, coexisting ions, and arsenic concentrations were studied in batch tests. Studies on the kinetic adsorption of MICB, the recovery of arsenic by the desorption solution, and the reuse of MICB were also carried out. The practicality and efficiency of an MCIB-packed column on arsenic removal were evaluated in a continuous system on industrial arsenic-containing wastewater discharged during the manufacture of GaAs supports. The results indicate that MICB favor the adsorption of both As(V) and As(III). The optimal pH value for As(III) and As(V) removal was 5. The adsorption of arsenic on the MICB is most likely an exothermic reaction. The effect of coexisting ions was varied and depended on their concentrations and species. The optimal desorption solution for arsenic recovery was 1M H2SO4, which resulted in a 95% efficiency for As(III) and 99% for As(V). In the continuous tests, the MICB-packed column exhibited excellent arsenic removal from wastewater without any pretreatment. These results provide strong evidence of the potential of MICB for removing As from industrial wastewaters.     

Response surface optimization of the removal of nickel from aqueous solution by cone biomass of Pinus sylvestris

Response surface methodology was applied to optimize the removal efficiency of Ni(II). Pinus sylvestris ovulate cones were used in this study. A 2(3) full-factorial central composite design was employed for experimental design and analysis of the results. The initial Ni(II) concentration (10-30 mg/l), pH (2.5-6.5) and biomass concentration (5-25 g/l) were the critical components of the removal optimized. The optimum pH, m (biomass concentration) and C0 (initial Ni(II) concentration) were found to be 6.17, 18.8 g/l and 11.175 mg/l, respectively. Under these conditions, removal efficiency of Ni(II) was 99.91%.     

Removal of chromate anions from aqueous stream by a cationic surfactant-modified yeast

The removal of chromate anions (CrO(4)(2-)) from aqueous solution by a cationic surfactant-modified yeast was studied in a batch system. Cetyl trimethyl ammonium bromide (CTAB) was used for biomass modification; it substantially improved the biosorption efficiency. The influences of solution pH, CrO(4)(2-) anion concentrations and biomass concentration on the biosorption efficiency were investigated. The biosorption of chromate anions by modified yeast was strongly affected by pH. The optimum pH for biosorption of CrO(4)(2-) by modified yeast was 4.5-5.5. Zeta potential values of modified and unmodified yeast were determined at various pH values. Concentrations ranging from 5.2 to 208 mg/l Cr(VI) were tested and the biosorptive removal efficiency of the metal ions from aqueous solution was more than 99.5%. Freundlich and Langmuir isotherms were used to evaluate the data and the regression constants were determined.     

Characterization of Pb<Superscript>2+</Superscript> biosorption from aqueous solution by<Emphasis Type="Italic"> Rhodotorula glutinis</Emphasis>

The yeast Rhodotorula glutinis was examined for its ability to remove Pb(2+) from aqueous solution. Within 10 min of contact, Pb(2+) sorption reached nearly 80% of the total Pb(2+) sorption. The optimum initial pH value for removal of Pb(2+ )was 4.5-5.0. The percentage sorption increased steeply with the biomass concentration up to 2 g/l and thereafter remained more or less constant. Temperature in the range 15-45 degrees C did not show any significant difference in Pb(2+ )sorption by R. glutinis. The light metal ions such as Na(+), K(+), Ca(2+), and Mg(2+) did not significantly interfere with the binding. The Langmuir sorption model provided a good fit throughout the concentration range. The maximum Pb(2+ )sorption capacity q(max) and Langmuir constant b were 73.5 mg/g of biomass and 0.02 l/mg, respectively. The mechanism of Pb(2+) removal by R. glutinis involved biosorption by direct biosorptive interaction with the biomass through ion exchange and precipitation by phosphate released from the biomass.     

Arsenic removal in an iron oxide-coated fungal biomass column: analysis of breakthrough curves

Column studies were conducted, using iron oxide-coated Aspergillus niger biomass, to examine the removal of arsenic [As(III) and As(V)] from an aqueous solution. The Thomas and Yan models were examined to predict the breakthrough curves. The Yan Model described the data better (based on the R(2) values) when compared with the Thomas Model. The adsorption capacity of the iron oxide-coated biomass estimated by the Thomas Model {1070 microg/g for As(V) and 700 microg/g for As(III)} was comparable to the calculated value of its adsorption capacity {1080 microg/g for As(V) and 880 microg/g for As(III)}.     

Removal of chromium from industrial waste by using eucalyptus bark

Several low cost biomaterials such as baggase, charred rice husk, activated charcoal and eucalyptus bark (EB) were tested for removal of chromium. All the experiments were carried out in batch process with laboratory prepared samples and wastewater obtained from metal finishing section of auto ancillary unit. The adsorbent, which had highest chromium(VI) removal was EB. Influences of chromium concentration, pH, contact time on removal of chromium from effluent was investigated. The adsorption data were fitted well by Freundlich isotherm. The kinetic data were analyzed by using a first order Lagergren kinetic. The Gibbs free energy was obtained for each system and was found to be -1.879 kJ mol(-1) for Cr(VI) and -3.885 kJ mol(-1) for Cr(III) for removal from industrial effluent. The negative value of deltaG0 indicates the feasibility and spontaneous nature of adsorption. The maximum removal of Cr(VI) was observed at pH 2. Adsorption capacity was found to be 45 mg/g of adsorbent, at Cr(VI) concentration in the effluent being 250 mg/l. A waste water sample containing Cr(VI), Cr(III), Mg, and Ca obtained from industrial unit showed satisfactory removal of chromium. The results indicate that eucalyptus bark can be used for the removal of chromium.     

Monitoring the denitrification of wastewater containing high concentrations of nitrate with methanol in a sulfur-packed reactor

Biological denitrification of high nitrate-containing wastewater was examined in a sulfur-packed column using a smaller amount of methanol than required stoichiometrically for heterotrophic denitrification. In the absence of methanol, the observed nitrate removal efficiency was only about 40%, and remained at 400 mg NO(3)(-)-N/l, which was due to an alkalinity deficiency of the pH buffer and of CO(2) as a carbon source. Complete denitrification was achieved by adding approximately 1.4 g methanol/g nitrate-nitrogen (NO(3)(-)-N) to a sulfur-packed reactor. As the methanol concentration increased, the overall nitrate removal efficiency increased. As influent methanol concentrations increased from 285 to 570, 855, and 1,140 mg/l, the value of Delta mg alkalinity as CaCO(3) consumed/Delta mg NO(3)(-)-N removed increased from -1.94 to -0.84, 0.24, and 0.96, and Delta mg SO(4)(2-) produced/Delta mg NO(3)(-)-N removed decreased from 4.42 to 3.57, 2.58, and 1.26, respectively. These results imply the co-occurrence of simultaneous autotrophic and heterotrophic denitrification. Sulfur-utilizing autotrophic denitrification in the presence of a small amount of methanol is very effective at decreasing both sulfate production and alkalinity consumption. Most of methanol added was removed completely in the effluent. A small amount of nitrite accumulated in the mixotrophic column, which was less than 20 mg NO(2)(-) -N/l, while under heterotrophic denitrification conditions, nitrite accumulated steadily and increased to 60 mg NO(2)(-) -N/l with increasing column height.     

Biosorption of Cr (VI) from aqueous solution by Rhizopus nigricans

The study was aimed to quantify the Cr sorption ability of powdered biomass of Rhizopus nigricans at the best operating conditions. The influence of solution pH, agitation, Cr (VI) concentration, biomass dosage, contact time, biomass particle size and temperature were studied. The optimum pH for biosorption of Cr (VI) was found to be 2.0. Higher adsorption percentage was noted at lower initial concentrations of Cr ions, while the adsorption capacity of the biomass increased with increasing concentration of ions. Optimum biomass dosage was observed as 0.5% (w/v). More than 75% of the ions were removed within 30 min of contact and maximum removal was obtained after 8 h. Biomass particles of smaller size (90 microm) gave maximum adsorption (99.2%) at 100 mg/l concentration. The adsorption capacity increased with increase in temperature and agitation speed and the optimum were determined as 45 degrees C at 120 rpm. Freundlich and Langmuir isotherms were used to evaluate the data and the regression constants were derived. The adsorption rate constant values (Kad) were calculated for different initial concentration of Cr ions and the sorption was found to be higher at lower concentration (100 mg/l) of metal ion.     

Equilibrium characterization of the As(III)-cysteine and the As(III)-glutathione systems in aqueous solution

Some arsenic compounds were the first antimicrobial agents specifically synthesized for the treatment of infectious diseases such as syphilis and trypanosomiasis. More recently, arsenic trioxide has been shown to be efficient in the treatment of acute promyelocytic leukemia. The exact mechanism of action has not been elucidated yet, but it seems to be related to arsenic binding to vicinal thiol groups of regulatory proteins. Glutathione is the major intracellular thiol and plays important roles in the cellular defense and metabolism. This paper reports on a study of the interactions between arsenic(III) and either cysteine or glutathione in aqueous solution. The behavior observed for the As(III)-glutathione system is very similar to that of As(III)-cysteine. In both cases, the formation of two complexes in aqueous solution was evidenced by NMR and electronic spectroscopies and by potentiometry. The formation constants of the cysteine complexes [As(H(-1)Cys)(3)], log K = 29.84(6), and [As(H(-2)Cys)(OH)(2)](-), log K = 12.01(9), and of the glutathione complexes [As(H(-2)GS)(3)](3-), log K = 32.0(6), and [As(H(-3)GS)(OH)(2)](2-), log K = 10(3) were calculated from potentiometric and spectroscopic data. In both cases, the [As(HL)(3)] species, in which the amine groups are protonated, predominate from acidic to neutral media, and the [As(L)(OH)(2)] species appear in basic medium (the charges were omitted for the sake of simplicity). Spectroscopic data clearly show that the arsenite-binding site in both complexes is the sulfur atom of cysteine. In the [As(L)(OH)(2)] species, the coordination sphere is completed by two hydroxyl groups. In both cases, arsenic probably adopts a trigonal pyramidal geometry. Above pH 10, the formation of [As(OH)(2)O](-) excludes the thiolates from arsenic coordination sites. At physiological pH, almost 80% of the ligand is present as [As(HL)(3)].     

Removal of Pb2+ and Ni2+ by bio-sludge in sequencing batch reactor (SBR) and granular activated carbon-SBR (GAC-SBR) systems

Living bio-sludge from domestic wastewater treatment plant was used as adsorbent of heavy metals (Pb(2+), Ni(2+)) and its adsorption capacity was about 10-30% reduced by autoclaving at 110 degrees C for 10 min. The living bio-sludge acclimatized in synthetic industrial estate wastewater (SIEWW) without heavy metals showed the highest Pb(2+) and Ni(2+) adsorption capacities at 840+/-20 and 720+/-10 mg/g bio-sludge, respectively. The adsorbed Pb(2+) and Ni(2+) were easily eluted (70-77%) from bio-sludge by washing with 0.1 mol/l HNO(3) solution. The heavy metals (Pb(2+), Ni(2+)) removal efficiency of both SBR and GAC-SBR systems were increased with the increase of hydraulic retention time (HRT), or the decrease of organic loading. The SBR system showed higher heavy metals removal efficiency than GAC-SBR system at the same organic loading or HRT. The Pb(2+), Ni(2+), BOD(5), COD and TKN removal efficiencies of GAC-SBR system were 88.6+/-0.9%, 94.6+/-0.1%, 91.3+/-1.0%, 81.9+/-1.0% and 62.9+/-0.5%, respectively with industrial estate wastewater (IEWW) with 410 mg/l glucose, 5 mg/l Pb(2+) and 5 mg/l Ni(2+) under organic loading of 1.25 kg BOD(5)/m(3) d (HRT of 3 days). The bio-sludge quality (sludge volume index: SVI) of the system was less than 80 ml/g. The excess sludge from both SBR and GAC-SBR systems with SIEWW under the organic loading of 1.25-2.50 kg BOD(5)/m(3) d contained Pb(2+) and Ni(2+) at concentrations of 240-250 mg Pb(2+)/g bio-sludge and 180-210 mg Ni(2+)/g bio-sludge, respectively.     

Removal of Arsenic(III) from Waste Water Using Lactobacillus acidophilus

Lactobacillus acidophilus was used for the removal of As(III) from 50–2000 ppb As(III)-containing water solution. Biosorption of As(III) by L. acidophilus was dependent on concentration (50 to 2000 ppb) and time (0 to 3 h).L. acidophilus(1 mg dry wt/ml) was able to remove 30, 60, 300, 420, 600 ppb As(III) from 50, 100, 500, 1000, and 2000 ppb of As(III)-containing water solution, respectively, within 3 h at pH 7. Moreover, by increasing the biomass of L. acidophilus(2 mg dry wt/ml) removal of As(III) was enhanced 1.66, 1.33, 1.16, 1.42, and 1.33 times, respectively. Fourier transform infrared (FTIR) and electron spectroscopy for chemical analysis (ESCA) spectrum of As(III)-loaded biomass was also investigated. An FTIR sample spectrum of L. acidophilus fresh biomass and As(III)-loaded biomass showed band stretching of fresh and As(III)-loaded biomass for O-H, 3423.43 to 3385.04 cm^?1, and for C-O, 1742.82 to 1731.14 cm^?1, and signified that –OH and –CO groups were also involved in the removal of As(III) from As(III)-containing water solution.     

Arsenic removal from waters by bioremediation with the aquatic plants Water Hyacinth (Eichhornia crassipes) and Lesser Duckweed (Lemna minor)

In this study the removal of arsenic by the Water Hyacinth (Eichhornia crassipes) and Lesser Duckweed (Lemna minor) was monitored under a concentration of 0.15mgL(-1) of the element. Plant densities were 1kg/m2 for Lesser Duckweed and 4kg/m2 for Water Hyacinth on a wet basis. The arsenic was determined in foliar tissue and water samples by hydride generation atomic absorption spectroscopy. The element was monitored as a function of time during 21 days. No significant differences were found in the bioaccumulation capability of both species. The removal rate for L. minor was 140mg As/had with a removal recovery of 5%. The Water Hyacinth had a removal rate of 600mg As/had and a removal recovery of 18%, under the conditions of the assay. The removal efficiency of Water Hyacinth was higher due to the biomass production and the more favorable climatic conditions. This specie represents a reliable alternative for arsenic bioremediation in waters.     

Phytoremediation of arsenic in submerged soil by wetland plants

Wetland aquatic plants including Canna glauca L., Colocasia esculenta L. Schott, Cyperus papyrus L. and Typha angustifolia L. were used in the phytoremediation of submerged soil polluted by arsenic (As). Cyperus papyrus L. was noticed as the largest biomass producer which has arsenic accumulation capacity of 130-172 mg As/kg plant. In terms of arsenic removal rate, however, Colocasia esculenta L. was recognized as the largest and fastest arsenic remover in this study. Its arsenic removal rate was 68 mg As/m2/day while those rates of Canna glauca L., Cyperus papyrus L. and Typha angustifolia L. were 61 mg As/m2/day, 56 mg As/m2/day, and 56 mg As/m2/day, respectively. Although the 4 aquatic plants were inferior in arsenic accumulation, their high arsenic removal rates were observed. Phytostabilization should be probable for the application of these plants.     

Removal of Pb2+ from aqueous system by live Oscillatoria laete-virens (Crouan and Crouan) Gomont isolated from industrial effluents

Cyanobacteria have been found to be potential biosorbents of metal ions from waste water. The Pb(2+) removal capacity of growing cells of indigenous cyanobacterium Oscillatoria laete-virens (Crouan and Crouan) Gomont was studied under batch experiments and it was found capable of removing Pb(2+) of lower concentrations (below 100?mg L(-1)). The effects of different concentrations of Pb(2+), on the growth rate of alga were also evaluated. The research parameters include the pH of the solution, contact time, initial concentration of Pb(2+), and culture density. Of the parameters studied, the pH of the solution was found to be the most crucial. The removal of Pb(2+) peaked at an initial pH of 5. The data obtained from the equilibrium experiments were found well fitting with the Langmuir isotherm with a maximum sorptive capacity (q (max)) of 20.36?mg?g(-1), indicating a good biosorbtive potential of growing cells. This was confirmed using scanning electron microscope and energy dispersive X-ray analysis, which showed the adsorption of lead on the surface of the cell. The species could tolerate a concentration as high as 60?mg L(-1) of Pb(2+). It was observed that the removal obeyed the pseudo-second-order kinetics. The percentage removal was found to decrease with increasing metal concentration, from 10 to 100?mg L(-1). FTIR analysis indicates the involvement of amino, carboxylic and amide groups in the sorption process. Among the desorbing agents evaluated, an efficient recovery of 90.2?% was achieved by HCl, in 24?h. Thus Oscillatoria laete-virens (Crouan and Crouan) Gomont seems to be a promising metal biosorbent for the treatment of Pb(2+), in waste waters.     

Use of X-ray absorption spectroscopy and biochemical techniques to characterize arsenic uptake and reduction in pea (Pisum sativum) plants.

Arsenite (As(III)) and arsenate (As(V)) uptake by peas was investigated using inductively coupled plasma/optical emission spectroscopy (ICP-OES) at pH below 4 and at pH 5.8. Additionally, total amylolitic activity and alpha-amylase (1,4-alpha-d-glucan glucanohydrolase; EC 3.2.1.1) activity was assayed in plants exposed to arsenic treatments. At pH below 4, the uptake for As(III) and As(V) in roots was 137 and 124 mg As kg(-1) dry weight (d wt), respectively. Translocation of arsenic to the aerial part was relatively low ( approximately 5mg As kg(-1) d wt). The uptake for As(III) and As(V) in roots at pH 5.8 was about 43 and 30 mg As kg(-1) d wt, respectively, and translocation of As to the aerial part was not detectable. None of the arsenic treatments affected the total amylolitic activity in roots; however, the shoots from all treatments showed an increase in the total amylolitic activity. Alpha-amylase activity in the pea leaves was not significantly affected by arsenic treatments. X-ray absorption spectroscopy (XAS) studies showed a reduction of As(V) to As(III) in the roots. From linear combination X-ray absorption near edge structure (LC-XANES) fittings, it was determined that arsenic was present as a mixture of As(III) oxide and sulfide in pea roots.     

Arsenic transport by the human multidrug resistance protein 1 (MRP1/ABCC1). Evidence that a tri-glutathione conjugate is required

Inorganic arsenic is an established human carcinogen, but its metabolism is incompletely defined. The ATP binding cassette protein, multidrug resistance protein (MRP1/ABCC1), transports conjugated organic anions (e.g. leukotriene C(4)) and also co-transports certain unmodified xenobiotics (e.g. vincristine) with glutathione (GSH). MRP1 also confers resistance to arsenic in association with GSH; however, the mechanism and the species of arsenic transported are unknown. Using membrane vesicles prepared from the MRP1-overexpressing lung cancer cell line, H69AR, we found that MRP1 transports arsenite (As(III)) only in the presence of GSH but does not transport arsenate (As(V)) (with or without GSH). The non-reducing GSH analogs L-gamma-glutamyl-L-alpha-aminobutyryl glycine and S-methyl GSH did not support As(III) transport, indicating that the free thiol group of GSH is required. GSH-dependent transport of As(III) was 2-fold higher at pH 6.5-7 than at a more basic pH, consistent with the formation and transport of the acid-stable arsenic triglutathione (As(GS)(3)). Immunoblot analysis of H69AR vesicles revealed the unexpected membrane association of GSH S-transferase P1-1 (GSTP1-1). Membrane vesicles from an MRP1-transfected HeLa cell line lacking membrane-associated GSTP1-1 did not transport As(III) even in the presence of GSH but did transport synthetic As(GS)(3). The addition of exogenous GSTP1-1 to HeLa-MRP1 vesicles resulted in GSH-dependent As(III) transport. The apparent K(m) of As(GS)(3) for MRP1 was 0.32 microM, suggesting a remarkably high relative affinity. As(GS)(3) transport by MRP1 was osmotically sensitive and was inhibited by several conjugated organic anions (MRP1 substrates) as well as the metalloid antimonite (K(i) 2.8 microM). As(GS)(3) transport experiments using MRP1 mutants with substrate specificities differing from wild-type MRP1 suggested a commonality in the substrate binding pockets of As(GS)(3) and leukotriene C(4). Finally, human MRP2 also transported As(GS)(3). In conclusion, MRP1 transports inorganic arsenic as a tri-GSH conjugate, and GSTP1-1 may have a synergistic role in this process.