Microbial biomass: An economical alternative for...

Indian Journal of Experimental Biology Vol. 41 , September 2003, pp. 945-966

Microbial biomass: An economical alternative for removal of heavy metals from waste water

Rani Gupla* & Harapriya Mohapatra

Department o f Microbi ology, Uni versity of Delhi South Campus, Benito Juarez Road, Ncw Delhi 110021. India

Today indi sc riminate and uncontrolled di schargc of metal contaminated industrial effluents into the environmclll has bccome an issue of major concern . Heavy metals, being non-biodegradab lc and persistent , beyond a permissible concentration form unspec ifi c compounds inside the cells thereby causing cellular tox icity. Thc only alternative to rcmove them from the wastewater is hy immobi li zing them. The conve ntional methods adopted earlier for thi s purpose included chemical prec ipitation, oxidation, rcducti on, filtrati on, electrochemi cal treatment, evaporation , adsorption and ion-exchangc resins. These methods require hi gh energy inputs especiall y when it refers to dilute solu tions. Here microbial biomass offcrs an economical option for remov ing heavy mctal s by the phcnomenon of biosorption. Non-li ving or dead biomass sequestcr metal (s) on thcir ce ll su rface duc to certain reactivc groups available like carboxy l, ami ne. imidazole, phosphate, sulph ydryl, su lfate and hydroxy l. The process can be madc economical by procuring spcnt biomass from industry or naturall y ava il able bulk biomass. A batch or a cOlll inuous process o r removal of heavy metal s dircctly from effluent s can be developed in a fi xed bcd reac tor using the immobi li zed biomass. Further biosorpti on potential of the biomass can be improved by vari ous physical and chemica l treatmcnts. Thc avai lability of variety of microbi al biomass and their metal binding poteilli al makes it a cconomical and sustainable option for developi ng effluent treatment process fo r remova l and recovery of heavy metal s.

Keywords: Effluent treatment , Heavy metals , Metal binding potential , Metal pollutant , Microbial biomass

A sudden boost in the industrial activities has contributed quantitatively as well as qualitative ly to the alarming increase in the di scharge of metal pollutants into environmental sink , especially the aqueous environment. Di spers ion of the metal ions in water bodies leads to their bi omagnification through the food chain and results in increased toxicity. This fact renders the removal of heavy metal s from aqueous sol utions indispensable. Thus, the boon of affl uence has in turn given ri se to the curse of effluent. More than fifty percent of the heavy metal pollution has been accounted to the anthropogenic act ivities. The tragic episodes of ' Itai- Itai ' and 'Minamata ' brought into focus the global concern regarding the impact of environmental pollution on human health . Since then several centers all over the world have been engaged in the deve lopment of processes for removal of heavy metals t

•4

.

The conventional processes used for effluent treatment are precipitation as hydroxides/sulphides, ox idation/reduction , and Jon exchange. These

* Author for correspondence: Phone: +9 1-01 1-61 11 933 Fax: +9 1-0 I 1-6885270 E-mai l: microzyme@ 123 india.com

processes are expensive especially when the contaminant metals are dissolved in large volumes and appear in the concentration range 10-100 ppm l

.

Moreover these are not ecofriendly in nature and result in the production of large amount of sludge. As a result an aquatic problem is transformed into a olid waste di sposal problem. Therefore, amo ngst the chemical adsorbent o nly ion exchange resi ns were considered as the option for remediation with least ecological problems. However, chemical res in s are expensive and the increasing demand for eco-fri endly technologies has led to the search of low cost alternatives that could be considered as single use materials. In thi s li ght, biological material s have emerged as an ecofriendly and economic option6

. For a long time acti vated carbon and peat occupied the place of prominence among biosorbent' s but since they were geographically restricted in distribution microbial biomass became the unanimous choice.

Microbial biomass can bind heavy metal s e ither actively or pass ively or by a combination of both processes7

. The passive phenomenon of ' biosorpti on' has several advantages over the active pheno menon of 'b ioaccumulation' . Growth physiology of an organism varies drastically with variations in the effluent compositio ns making it difficult to express

946 INDIAN J EXP BIOL, SEPTEMBER 2003

mathematicall l -IL.. On the other hand, biosorption involves use of non-growing biomass/dead biomass for sequestration of the metals, thus the process is independent of metabolic activity. Another major disadvantage of bioaccumulation is recovery of the accumulated metal by destructive means whereas in biosorption desorption is accomplished by simple phys ical methods without damaging the biosorbent's structural integriti I. Moreover, biosorption has an edge over bioaccumulation with easy and cost effective procurement of the biomass either as a byproduct of a large scale fermentation process or bulk procurement from natural water bodies I. Last but not the least being a surface phenomenon, most of the biosorption is generally completed within few min of contact with the biomass.

Several workers l.3-5.12- 15 have presented exhaustive

reviews on the biosorption.

Microbial biomass: taxonomic considerations A wide variety of biological materials have been I . d f h ' I b' . .. I 1617 exp OIte or t elr meta IOsorptlon capacities' . .

One can begin the count down from the classical use of activated carbon to peat and end up with the

'd d f . b' I b' b . I 1819 WI esprea repOits 0 mICro Ia losor ent matena s . . Fungi, yeast and bacteria as by-products of industrial fermentation processes while macro algae due to bulk availability of their biomasses from natural water bodies have attracted most of the attention as is evident from the literature2o-25 .

Fungi and yeasts belonging to the genera Rhizopus, Aspergillus, Penicillium and Saccharomyces have shown excellent metal biosorption capacities26-3o. Muraleedharan et al.30have screened several tropical wood rotting mushrooms for Cu biosorption. Ganodenna lucidum have shown good sorption potential for Cu and rare earth metals31. Karavaiko et al.32 have reported selective extraction of noble metals from solutions by microfungi, yeasts and actinomycetes. Uranium biosorption potential has been reported for polyester entrapped foam beads of Trichoderma harzianum33. Among bacteria, Bacillus subtilis has been identified to have potential for metal sequestration and has been used in the commercial biosorbent preparation3

.26. Sahoo

et al. 34 have reported good Cu and Cd removal potenti al for Bacillus circulans. Besides, there also exist reports on the biosorption potential of Pseudomonas sp. , Citrobacter sp., Zoogloea ramigera, Arthrobacter and Streptomyces spp.

Though available as a cheap industrial by-product, one major disadvantage of using biomasses from industrial processes is the presence of impurities due to the adhering fermentation broth residue that might interfere with its metal sorption capaciti 5

. Dias et al. 40 have compared the biosorptive potential of waste biomasses obtained from four different distilleries. They observed marked varia tion in metal binding capacIties of these biomasses. These variations could be attributed to diversity in the microflora composition and culture conditions in each of these production media. Omar et al.4 1 in their studies on uranium removal by the brewery yeast Saccharomyces cerevisiae observed higher U removal by unwashed, dried and ground yeast as compared to its washed and live counterpart. Waste products of antibiotics industries were able to remove 99% Zn and Cd and 95 % Ni from the metal solutions25 . Further, in case of bacteria, immobilization of the biomass prior to its application in continuous columns/fixed bed reactors is must. Exhaustive lists of the metal sorption capacities of heterotrophs have been presented by3.13.42. Mc Lean and Beveridge43have reviewed the metal binding capacity of bacterial surfaces.

Besides heterotrophs, photoautotrophs especially seaweeds have been extensively worked upon . The brown seaweeds belonging to the gener(} Ascophyllum and Sargassum form the most exploited organisms for h . h ' hI' .. ? I 22 44 45 Th h t elr Ig meta sorptIOn capacltles-' ". e ot er

exploited groups include Fucus versiculosus, Eisenia, Laminaria, Spirulina , Porphyra, Cyanidium. Among the green algae the genus Chlorella has been identified as a potential candidate for metal biosorption46-48 . Gale49 has reported effective removal of Pb by blooms of Chlo relia , Cladophora, Rhizoclonium, Hydrodictyon, Spirogyra and Oscillatoria. Kuyucak and Voleskyll have evaluated gold and cobalt removal abilities of green (Cosium taylori, Halimeda opuntia), brown (Macrocystis pyrifera, Undaria sp., Laminaria sp. Ascophyllum nodosum, Sargassum jluitans, S. natal1s) and red (Chondrus crispus, Palma ria palmate, Porphyra tenera) marine algal species.

Though the potential of these algal cultures to remove heavy metals under laboratory conditions have been well established, the major bottleneck lies in their commercialization and large-scale production . Recent developments in algal biotechnology have resulted in the commercial production of certain strains for their use either as animal feeds, chemicals ,

GUPTA & MOHAPATRA : MI CROBIAL BIOMASS FOR REMOVAL OF HEAVY META LS 947

biochemicals or fertilizers or as a source of food for humansso. The commercia lly grown genera are limited in number (only 8 genera) and include Spirulina, Chlorella, Scenedesmus, Phaeodactylum, Botryococcus, Chlamydomonas, Dunaliella and Porphyridium . Of these, only Spirulina and Chlorella have been produced commerc iall y, exclusive ly for their metal removal capacitiesso. Unlike fungi, yeast and bacteri a, the commercia li zed a lgal group has the advantage of be ing a better defin ed materi a l as the conditions in the natural water do not change as much as those of the culture medi a.

Above rev iew of the lite rature reveals that apart from few genera such as Spirulina, Nostoc , Cymodocea and Scenedesmus the re ex ist very few reports of cyanobacteri a in the area of biosorption

s,-ss .

Developing biosorbents for commercial application: Immobilization and reinforcement

Despite the tremendo us potentia l of the a lga l cell s for heavy metal remo val they still face major challenges for be ing expl oited commercia ll y . Commercial biosorbents need to fulfill a number o f criteri a such as: (i) hi gh biosorption capac ity at equilibrium i. e. they should contain as little as poss ible of inert materi al in the ir binding sites (ii ) fa vorable biosorptio n kinetics i.e. particles should be hydrophilic and poro us in nature (iii ) maintenance of smooth fl ow dynamics in a reactor - thi s prevents the use o f either very small or strong ly swelling particles in the column (iv) amenable to regeneration - thi s necess itates desorption by minimal poss ible vo lume of desorbing agent w ithout damaging the bi osorbent (v) good mechanical strength (v i) temperature stability and (v ii ) res istance to chemi calss6

.

The tendency of the free algal ce ll s to clump together leads to clogging of the column. This al so necess itates excess ive hydrostati c pressure to generate suitable flow rates. Moreover, the frag ile nature of the free algal cell s renders them susceptible to disintegration due to high pressures. All these limitations make it imperative to immobilize these into suitable matrices prior to the ir use as commercial biosorbents in columnss7

. Not all algae require immobilization; macro algae such as Sargassum or Chara need only proper sizing prior to use while all micro algae require some degree of immobili zation or pre-treatment or hardening. Immobili zation o f the nonviable cell s has been carri ed out in several matrices natural as well as synthetic . Table 1 li sts the use of various immobilizing matrices fo r metal bi osorption.

Among the most commonly used matrices are polyacrylamide, calcium/sodium alg inate and silica. Other matrices may inc lude sta inless steel wire, pan scourers, cotton webbing, alumina, coal, foa ms or po lyviny l chloride. Philip et al. 36 have reported the use of several commonly ava ilable, inex pensive matlices for immobilization of Pseudomonas aeruginosa cells. These included a lumina, coconut she ll , g iridih bituminous coal, g lass, ceramic materi a l, refractory bricks, rice husks, silica gel and sand . S ilica immobili zed matrices posses' greater mechanical strength coupled with excellent fl ow characte ri stics . The co mmercial alga l biosorbent AlgaSORB™ developed by Bio-Recovery Systems, Inc., utili zes silica as the immobili zation matri x7s

. Higher metal removal abilities by immobili zed organisms have been reported by severa l workers 70.70. Aspe rg illus niger myceli a immobili zed in po lyurethane foam showed three-fold inc rease in Cu removal as compared with that o f free cells7o

. . 38 7? 73 . 7' Res rns such as po lysul fo ne . -, and epoxy res Ins -

have also been successfull y used fo r entrapment and immobilization. Polyvinyl alcohol (PV A) immobi li zed beads of Phormidium valderium were able to remove 80% and 60% Co per gram in 24 hr60 .

Se lecti on of matrices should be done such that they do not hamper or slow down the sorption capacity o f the bi omass by blocking or consuming the bi nd ing sites. Park e f al.39 observed a decrease in biosorpti on fo llowing immobili zatio n of Zoogloea ramigera cell s in calcium a lginate. Park et al.59 have a lso reported lower Cd uptake by Ca-a lginate encapsulated Aureobasidium pullulans than by the free biosorbent.

Apart fro m immobili zation, re inforcement and cross-linking also provides a suitable means of stabili zing the biosorbent. Cross-linking can be achieved by formaldehyde, glutera ldehyde and/or polyethylene imine. Higher durability was reported for cross-linked cell s (tri ethy lene tetramine and g lutamic di aldehyde 1 % w/v) of Zoogloea ramigera39

.

The authors have reported the cross-linked capsules to maintain the ir mechanical strength and adsorpti on/ desorption capac ity even after 30 cycles of repeated use. Ashkenazy et al.77 have reported benzaldehyde in acetonitrile to be a good fi xati ve, which reta ined the initia l Pb biosorpti ve potenti al of Saccharomyces cerevisiae . Fixation with benzaldehyde in acetonitr ile a lso rendered the bio mass res is tant to mi crob ial spoil age fo r up to 1 year at 4°C.

Some workers have a lso reported the pre-treatment of the biosorbent w ith acids, alka li , salt solut ions,


detergents or organi c solvents to affect the biosorptive potential of the biosorbent2

1.78.79 . Drying of the biosorbent material has reported to have different effects on the biosorptive potential as evident form Table 2.

Process parameters affecting biosorption The biosorption process being a cell surface

phenomenon is to a large extent affected by the chemical compos ition of the cell surface. Apart from the variations in the chemical nature of the bi osorbent , the process is also affected by a variety of other physio-chemi cal parameters such as pH , temperature, initia l metal concentration , time of contact, co-ions and biosorbent concentration. A comprehensive deta il of the vari ous parameters affec ting the biosorptive capac ity of the biosorbents has been listed in Table 2.

pH p H of the environment influences the biosorption

process in several ways. The most important effect is

the change imparted by it on acti ve b inding s ites, which in biosorption are usually acidic in nature. Decrease in pH leads to their protonation thereby decreasing their negative charge and consequentl y the cation binding. On the other hand, an increase in p H increases the availabi lity of the negati ve ly charged free sites for electrostatic attraction of cati ons, thereby resulting in an increase in the cation binding capac it/ 5

. Sal' et al.35 have reported max imum sorption of Cu at pH 7 .0 whi le in case of Ni the trend continued till pH 8.0 . Vianna el al.89

have reported low binding of Cu , Cd and Zn at ac idic pH (2.5and 3.5) while the non-protonated biomass of Bacillus lenlus and Aspergillus oryzae showed higher Cd sorption , 80 and 20 mg/g, respecti ve ly. Puranik and Paknikar38 have reported pH optima of 4.5, 6 .0 and 6 .5 for Pb, Cd and Zn uptake, respectively by Citrobacler strain MCM B-1 8 1. A pH value of 5 .8 was found to be optimum fo r Ag sorption by

Table I - Immobili zati on matri ces/condit ioning used in metal biosorption

Immobil izati on matri x

Lyophi l ized biomass

Organism

Pseudomonas aeruginosa Aspergillus niger, Penicillium chrsogenum, Mucor miehei Pseudomonas aeruginosa

Calcium/Sod ium alginate Aureobasidium pullulans Zoogloea ramigera Tremetes versicolor

Polyv inyl alcohol Dry ing

Polyacrylamide

Silica

Polyurethane foam

Polysulfone

Epoxy res ins M agnetite (Fe)0 4)

Phormidium valderium Sargassllln sp. Cymodocea nodosa Sargasswn spp. GloeOlhece magna Azalia fi liculoides Rhizopus arrhizus

Chlorella regularis Streptomyces viridochrol1logenes Pseudomonas EPS 5028

TM Chlorella vulgaris (Al gaSORB )

Rhizopus oligosporus Aspergillus niger Rhi zo pu s a r rh i z u s

Bio-Fix (consortium) Phorl1lidiwn laminosum Citrobacter strai n MCM B - 181 Mu co r rOllxi i

Phormidiwn laminosw l1 Ellferobacler

Metals adsorbed References

U 37 U 58

Ni . Cu 35

Cd 59 Cd 39 Cd 23

Cd2+, Co2+, Cu2+, Ni2+ 60 Cd, Zn 61 Cu, Zn 62 Cd, Cu 22 Cd, Mn 63 Ni 64 Fe(CN),) 65

U 66 67 68

Au, Cu, Ni, U, Pb, Hg, Cd, 107 Zn, Ag

Cd 69

Cu 70.7 1

A I)+, Cd2+, Zn2+ and Mn2+ 3 72

Cu2+, Fe2

+, Ni2+ and Zn2+

Pb, Cd, Zn 38. 73

Cu2+, Fe2+, Ni2+, Zn2+ 72

Ni2+ 74

.J ~ ~ -..- -+

Table 2 - Effect of process parameters and biosorptive potential of some microbial biosorbents

Organism Time pH Temperature Biosorption Biomass Effect of Eluant Reference kinetics capacity pretreatments cations/anions

Aspergillus niger I hr 5.0 30°C 14-15 mg Cu2+/g Immobilization on EDTA (I mM) 70 polyurethane foam increased biosorption Cl

C

Chlorella vulgaris 6.8 3.1 mg Znlg 25 ~ » Sargassum fluitans 15 min 4.0 0.94 mmol/g Protonated and 21 Ro

crosslinked with s:: 0

fonnaldehyde :c »

Zoogloea ramigera 10-20 min 6.5 37°C 2.21 mg Cdlg (free Cross linking with HCI (1 M) 39 -0 »

cells); 1.78 mg 1% (w/v) triethylene -l ;:0

Cdlg (Ca alginate tetramine & 1% » immobilized); 1.11 gluteric solution s:: mg Cdlg (bead increased durability n entrapped) ;:0

0 Cll

Spirogyra sp. 2hr 2.0 18°C 14.7 mg Cdlg 80 ):: r

Sargassum sp. 1 to 6 6hr 4.5 22°C 0.9 to 0.66 mmol Protonation with HCI CaCI2 (1%), 22 to

Cdlg; 0.93 to (0.1 M) Ca(NOJh and (5 0.89 mmol Culg HCI (0.1 M) at s::

» SIL ratio 1.0 gIL en

en

Bacillus simplex 2hr 9.0 Cd >Ni ~Co >Sr EDTA (50 mM) 81 'Tl 0 ;:0

Cymodocea nodosa 20 min 4 .5 (Cu) 52.68 mg Culg; 62 ;:0 tTl

5.5 (Zn) 46.56 mg Znlg s:: 0

Citrobacter strain 5 min 4.5 (Pb) 28°C 23.6 mg Znlg; Several chemical Pb> EDTA (0.1 M) 38 < » MCM B -181 43.48 mg Cdlg; treatments increased Zn>Cu>Cd>Ni>Co at HCI (0.1 M) r

58.78 mg Pb/g Cd sorption but not pH4.5 0 'Tl

Zn and Pb. Biomass Zn>Cu>Cd>Ni>Co at :c could be immobilized pH 6.0 tTl » on polysulfone <

-< Rhizopus o/igosporus 20 mi n 5.0 30°C 126 mg Pb/g 82 s::

tTl

Streptomyces rimosus, 15 min 6.0 (Cu) More than 90% Penicillium biomass Zn >Cu>Ni>Ca>Na 83 -l »

Penicillium 7.0 (Zn) removal was acid treated r en

chrsogenum, 8.0 (Ni) Saccharomuces car/sbergensis, Saccharomyces cerevisiae

(- Collld) '-0 .p. '-0

\0 Ul

Table 2 - Conld 0

Organism Time pH Temperature Biosorption Biomass Effect of Eluant Reference kinetics capacity pretreatments cations/anions

Rhodymenia palmale, 5 min 2.0 to 6.0 Biosorption 40.11 mg Aulg by C. vulgaris could be Thiourea 20 Porphyra yezoensis, 2.0 to 6.0 by R.palmate; 83.5 mg immobili zed on si lica (0.1 M) pH 2.0 Laminaria japonica, 5 min 6.0 Chlorella Aulg by Eisenia bicyC/is, 2.0 to 4.0 pyrenoidosa C.caldarium; 70.68 Macrocystis pyrifera, 5 min 6.0 was highly mg Aulg by Cyanadium caldarium, 60 min 2.0 to 6.0 temperature S.platensis; Spirulina platensis, 5 min 2.0 to 6.0 dependent: 97 .6 mg Aulg by Chlorella pyrenoidosa, 5 min 2.0 to 6.0 increased c.pyrenoidosa Chlorella vulgaris 5 min with

5 min increase in 5 min temperature

with a Z maxima at 0

60°C. :; z

Saccharomyces 15 min 4.0-6.0 4°C 104.8 /lmol Aglg Pretreatment with Cd & methionine 84 cerevisiae NaOH & SOS decreased Ag sorption; tTl

X decreased sorption Na+ & SO/ ' at a "0

OJ concentration 1000 0 fold excess that of Ag .r had slight inhibitory Vl

effect. tTl

~ Rhizopus spp. Based 7.0 30°C 190 mg Co/g HCI and 85

tTl 3::

biosorbent PFBI 2 H2SO. (0.1 N) OJ tTl

Cu2+, A1 3+, Fe3+, Fe2+ ;;0

Pseudomonas strain 10 min 5.0 541 mg Ulg 37 tv

(lyophilized cells); and Th4+ inhibited U 0 0 w

410 mg Ulg (live so~tion . CO/' and cells) SO ' at 1000 mdldm3

inhibited U sorption

Azalia filiculoides 20 min 2.0 Room 99.9% removal of 45 temperature Au

Rhizapus arrhizus IS min 6.0 to 7.0 Biosorption Biosorbent stable at cr, N03., SO" , C03. HN03 (2 M) 86,87 (U233 , capacity me~sured pH 2.0 and 11 .0 and CH3COO' (O.IM) Pu239), in terms of decreased Pu sorption

2.0 distribution ratio (Am24 1, U-3071, Pu-9653 , Cel44, Am-8196, Eu-7113, Pm147, Pm-9386, Ce-9448, Eu152- Zr-1995.

154, (-Contd) Zr95)

"1' ..J... ... .... ..,..

Organism Time pH Temperature kinetics

Trametes versicolor 60 min 5.0 to 6.0

Phanerochaete 2 hr 6.0 chrsosporium

Saccharomyces 60 min 4.5 Temperature cerevisiae independent

Rhizopus nigricans 30 min 2.0 45°C

Azolla filiculoides 10 min 6.5 18°C

Pseudomonas 10 min 7.0 (Cu), 37°C aeruginosa 8.0 (Ni) (lyophilized)

Table 2 - Contd

Biosorption Biomass capacity pretreatments

102.3 mg Cdlg (live Heat treated dead entrapped cells); entrapped cells 120.6 mg Cdlg showed higher (dead fungal biosorptive capacity mycelia)

23.04 mg Cdlg; 69.77 mg Pb/g; 20.23 mg Cu/g

2.4 rrunol UO/+/g Dry cells performed better than live cells

47 mg Cr/g

43.4 mg Nilg Alkali reconditioning improved adsorption capacity

265 mg Nilg, Ni sorption: Cd most 137.6 mg Cu/g antagonist; Fe, Cu,

Co and Pb caused some degree of inhibition; Cr or Zn had marginal effect; Na, K & Ca enhanced sorption.

Cu sorption: Cd most antagonist; Inhibited slightly by Zn, Extent of inhibition to the same extent by Fe & Co; Na, K&Ca increased sorption.

Effect of Eluant cations/anions

HCl (10 mM)

Affinity Cu>Pb>Cd

Slight inhibition HCl and observed in presence of Ca2+

H2S04 (0.2 N)

Pretreatment with HCl, H2SO4,

NaOH, N~OH& HN03 and toluene increased nitiloacetic metal sorption. Oven acid (0.1 N) ; heating (80°C), CaC03 (10 autoclaving, detergent mM) and acetone treatments decreased sorption

Reference

23

24

41

88

64

35

a C :::j > Ro 3:: 0 ::c > ...., > -l ::0 > 3:: n ::0 0 O:l :; r O:l

0 3:: > C/) C/)

'"r:I 0 ::0 ::0 tTl 3:: 0 < > r 0 '"r:I ::c tTl > < -< 3:: tTl -l > r C/)

'D VI


Saccharomyces cerevisiae84. Hi ghest speci fic lead

uptake by Rhizapus oligosporus was obtained at pH 5.082. Zakharova el a l?5have reported an increase in the degree of metal sorption with an increase in the solution pH for a number of microbial biosorbents (bacteria, fungi, yeast, ascomycetes and algae) tested. Optimum pH for removal of U by Saccharomyces cerevisiae was found to be 4.541. Optimum pH for biosorption of Cd, Pb and Cu by filamentous fungus Phanerochaele chrysosporium was found to be 6.024 .

Sar and D' Souza37 observed maximum U uptake by Pseudomonas aeruginosa at pH 5.0 while the sorption was negligible at lower p H. They attributed thi s firstl y to the high solubility of uranyl ions and competiti on by W for U binding sites in the ac idic condition, secondly increased binding affinity of the biomass for monovalent uranyl species rU0 20H+, (U02)\OHs)] fo rmed at higher pH (4.0-5.0) over the divalent (UO/+) at low pH (2.0) and thirdl y to a decrease in di ssolved uranyl ion concentrati on at p H above 5.0 due to fo rmati on of so lid scheopite (4UO,.9H20 ). Maximum biosorpti on of Cd ions by Ca-a lginate immob ili zed Trametes versicolor occulTed between pH 5.0 and 6.0. Beyond 6.0 there was a drast ic decrease in sorption , which could be attributed to the format ion of cadmium hydrox ide complex 23 . These references exemplify a second way by which the pH affects metal sorption. Certain meta ls occur in thei r free and hydrated form at lower p H and as their hydrox ides at hi gher pH. This leads to precipitation and thus red uced avai lability of the metal ions. In such cases it is important to consider several species of one metal ion as an individual sorbate. As sorpti on increases with decreasing solubility, hydrolyzed metal ions are eas ily sorbed than free metal ions. Less energy is required to re-o ri ent the hydrated water molecules of these less hydroph ilic meta l ions IS.

Still other metal ions such as Au, Cr, Hg, and Ag have a greater tendency of occ urring as negatively charged complexes e.g. AuCN', AuCI"4as in case of Au. Such metal ions either show a decrease uptake with increasing pH or remain insensitive to pH change. Maximum removal of gold by Azalia jllicilloides was observed at pH 2.045

. The authors have attributed thi s to the anionic nature of AUCI"4' Bai and Abraham88 observed increased sorption of Cr (V I) by Rhizopus nigricans biomass at pH 2.0. They attributed this to the predomi nance of anionic spec ies of Cr (HCr0"4, Cr20 \, Cr40 20 13 and Cr20 20 10) at thi s low pH, which could eas il y in teract with the positi vely charged cell wall li gands. Maximum Co

removal (190 mg/g) by Rhizapus spp. based biosorbent PFB I was obtained at p H 7.085 . They however observed an average biosorption of 85 % in the pH range 2 to 8.

Time kinetics The major advantage of the biosorptive metal

uptake is its fast equil ibrium kinetics. Biosorption generally completes within fi rst few min of the in itial contact of biomass with the metal bearing solution, Following thi s, equi librium is es tab li shed between the metal uptake by the biomass and the residual metal in the so lution. Rapidi ty of the process of biosorption seems to be consistent with the mechan ism of passive adsorption to the cell s rather than being a metabolica lly acti ve process4

.90

,

Blanco el al.72 have reported a relatively hi gher time of contact (60 min) wi th 38-65% recovery of heavy metals, viz. Cu, Fe, Ni and Zn by polysulfone and epoxy bead immobili zed Phonnidiul11 laminoslllll. Sal' and D'Souza37 have reported 90% of U load ing to occur within 10 min of contact with li ve Pseudomonas cells. Eq uili brium was reached with in 60 min of contact in case of li ve cell s while lyophili zed biomass showed much slower rate and reached satu rat ion onl y after 120 min. Majority of Cd sorpti on by free Aureobasidium pullulans biosorbent was establi shed within 10 min of exposure59 Gold removal by Azalla jlliculoides was rapid with nearly 80% of the metal being removed within 20 min of exposure at p H 2.045

, Compared to the above system U sorption by Pseudomonas aeruginosa takes a much longer time with 50% sorpti on tak ing place after I hr of ex posure36 and equilibrium being reached after 2 hr. Pb biosorption by Rhizapus oligosporus was very rapid during the initi al stages of sorption process (0-20 min) but reached equi li brium onl y after 14 hr of exposure82 . Time kinetic studies of Citobacter starin MCM B-1 81, revealed the metal uptake to be rapid with 85 % Pb, 70% Cd and Zn being adsorbed in the first 5 min 38 . Cadmium biosorption by Zoog loea ramigera reached equilibrium fo llowing 10-20 min of exposure39

. For all the micromycetes rested for their Ag sorption, highest rate of the process was observed during the first min of incubation reaching the equilibrium within 20 min91 . Bai and Abrahamg8

observed rap id adsorption of Cr ions during initi al 30 min of sorbent contac t in case of Rhizopus nigricans biomass. Omar et al.4 1 in their studi es on U removal by Saccharomyces cerevisiae observed max imum removal to occur during first 60- 100 min of

GUPTA & MOHAPATRA : MICROBIAL BIOMASS FOR REMOVAL OF HEAVY METALS 953

exposure. In contrary to the above reports, Say et al.24

reported more than 60% adsorption of Cd, Pb and Cu following an exposure for 2 hr 111 case of Phaenerochaete chrysosporium. Hi gh initi al Cd biosorption rates were observed for Ca-alginate immobili zed Trametes versicolor at the beginning, which approached equilibrium within 60 min of

23 exposure .

External metal concentration Initi al metal concentration plays an important ro le

in determining the biosorpti ve capacity of a biosorbent. Generall y, it has been observed that an increase in the initi al metal concentration results in an increase in the metal sorpti on capac ity of the biosorbent, which culminates in a pl ateau at very hi gh metal concentrati on. The metal sorption capacity of an organi sm touches its peak at these hi gh metal concentrations. At this point the sorption of the metal is limited by the ava il ability of the number of binding sites 111 the biomass . Thus, at low metal concentrations (as encountered in efflu ent samples) the biosorption capac ity of the biosorbent is not full y utili zed l5.

An increase in the specific metal uptake with increase in initial metal concentration was observed in case of Citrobacter strain MCM B_1 8 138 . More than 90% of gold was removed from solutions containing AuCl"4 in the range 2 to 10 mg/L with Azalia Jiliculoides4

'i . The max imum specific Pb sorption by Rhizapus o/igosporus increased with an increase in initial lead concentration up to 200 mg/L beyond which the system attained equilibrium82

. Similar has been reported fo r sil ver biosorpti on by mi cromycete91

.

In contrast to the above reports , S ai and Abraham88

have observed a decrease in percentage adsorpti on of Cr ions by Rhizapus nigricans with an increase in the initial metal concentration from 50 to 400 mg/L. However, they have reported an increase in the spec ific biosorption with an increase in ex tern al metal concentration.

Biosorbent concentration With an increase in the biosorbent co ncentration

there resul ts a corresponding increase in the total metal removal accompani ed by a decrease in the specific uptake. This is due to the fac t th at total adsorption is dependent upon the number of ava il able binding sites whereas specifi c uptake is calculated as the amount of metal adsorbed per we ight of the biosorbent l,92 .

Similar has been reported fo r sorption of Cu, Fe, Ni and Zn by immobili zed biomass of Phormidium laminosum72 . A reverse trend was observed in case of immobili zed li ve cells of Rhizopus oligosporus where there was an increase in the specifi c biosorption with increase in biomass69

. The authors however observed a decrease in the spec ifi c biosorpti on with increase in biosorbent concentrati on in case of free Rhizopus oligosporus cells.

A decrease in specific uptake by encapsu lated biosorbent with an increase in the loading capacity of the biosobent was reported for A ureobasidium pullulans59

. An opti mum biomass concentration of 5 mg/L resul ted in 99 .9% removal of gold by Azolla f iliculoides45

. Sil ver removal increased with biomass concentration at 1 mg/cm3 to 25% and at 8 mg/cm3 to 78 %84 . For all initi al lead concentrations studied , the maximum specific lead uptake decreased with increasing biosorbent concentrations2

. They obtai ned max imum lead uptake capacity of 126 mg/g with an initi al lead to biosorbent ratio of 750 mg/g. Simil ar results of decrease in specific metal uptake with increasing biomass concentrati on has also been reported for Pb, Cd and Zn sorption by Citrobacrer strain MCM S-1 8 1 38 . Zhou 93 observed a decrease in Zn biosorpti on with increase in biosorbent (Rhizopus arrhizus) part ic le size and its concentrati on. Bai and Abarh amR8 also reported similar trend fo r removal of Cr ions by Rhizapus nig ricans.

Presence of cations, anions and ligands

Cations Cations decrease the biosorpti on of a metal by

competing fo r the same metal bindjng sites. Thus, effect of cations on biosorpti on can lead to the constructi on of selecti vity of biosorption seri es based on biosorpti on of metals from a mi xture solution. For biosorption there exi sts no competiti on between hard and soft metals but competiti on among boderline metals occur due to simjlarities in their co-ordinati on h . 2

C emlstry . Metal sorption by Cilrobacter strain MCM B-1 8 1

biomass in presence of equimolar multimetal cations at pH 4.5 and 6.0 revealed a preferenti al order of metal sorpti on as Co < Ni < Cd < Cu < Zn < Pb at pH 4.5 and Co < Ni < Cd < Cu < Zn at pH 6.038

. Sar et al.35 have in ves tigated the effect of the presence of cocations on Ni and Cu sorpti on in Pseudomonas aeruginosa they have reported that the presence of Cr and Zn marginally decreased Ni sorpti on while ell

954 INDIAN J EXP BIOL. SEPTEMB ER 2003

sorption was onl y s lightly inhibited by Zn. Fe, Cu, Co and Pb caused some degree of inhibiti on in Ni sorp tion as was observed for Cu sorpti on in presence of Fe and Co. Cd was the most poten t inhibitor of Ni sorpti on as was Pb for Cu sorption . Sorption of both the meta ls was enhanced in presence of Na, K and Ca.

Radioacti ve e lements can be strongly binding and therefore radioacti ve and heavy meta l ions mutually infl uence each other's uptake. Sar and D 'Souza3

? in case of Pseudomollas strain observed a significant antagon ism in U sorpti on to be offered by Th , Fe, Al and C u whi le metals like Cd, Pb and Ag had no effec t.

Soft metal like Au , Ag have a cova lent binding tendency, the competition by e lec trostati c binding of other anions could not be severe for thi s metal. For competition in covalent binding the charge of the metal is irre levant. The level of co mpetition depends mainl y on whether two meta ls use the same binding s ites and how strong ly they are bound to any g iven site. The effect of competing io ns (Cd and Na) on si I ver sorption by Saccharomyces cerevisiae was st udied by Singleton and Simmons84

. They observed that Cd inhibited Ag+ sorption by 21.2% at 10 mmol/dm' while Na+ affected biosorpt ion when 1000-fold in excess o f Ag in soluti on. Say el al.24 observed lower co mpetitive biosorptio n capac iti es fo r Cd , Pb and Cu by Phaenerochaele chrysosporium as co mpared to those under non-competiti ve cond itions . The biomass fo ll owed the affinity seri es Cu > Pb > Cd.

Anions alld ligands Ani ons of the meta l sa lt balance the positive charge

of the meta l ion and occur in any meta l bearing so luti on. The most important effect of anions on biosorption is through the formation of complexes with metal ion s in soluti on. Whether thi s has a pos iti ve or negative effect o n the overall metal sorption depends on the level of affinity of the biomass for the complex in comparison to the free meta l cation . Greene el al. 94 observed the degree of gold sorption to be strong ly dependent o n the compet ing li gands present in the so luti on. The anions can also affec t meta l sorption process by binding to the ac ti ve site and changing their charge.

Puranik and Pakn ikar38 whil e investigating meta l sorption by Cilrobacler stra in MCM 8-1 8 1 observed a decrease in the range 0.7-15 % for meta l sorption in presence of equimolar concentration o f borate, carbonate, chloride, sulfate, nitrate and acetate while in presence of phosphate and c itrate the uptake of the meta ls was reduced in the range 24-86% . Among the

anions tested for the ir effect o n U sorption by Pseudomonas strai n, Sar and D 'Souza3

? observed 18% and 26% reduction in U uptake by for lyophilized and li ve ce lls, respect ive ly in presence of CO/". Zn sorption by Rhizopus arrhizus was reduced in presence of ligands chiefly due to formation of metal compl exes of less biosorbable nature in the seri es EDT A> SO ~ - > C1 .93

. Single ton and Simmons88

studied the effect of li gands (SO ~ - and methionine)

on sil ver sorption by Saccharomyces cerevisiae. They observed that methionine inhibited Ag+ sorpti on by ! 3.3% at 100 mmolldm3 while SO ~ affected bio

sorption when present in lOO-fold excess of Ag in the soluti on. Tobin et al .95 have reported the inhibitory seri es of anions for metal sorption by Rhizopus arrhizus biomass to fo llow the order EDTA »SO ;- >cr>

PO ~- >glutamate>CO i- .

Temperature Simple physical sorption is generally exothermic.

However this cannot be extrapo lated to the phenomenon of biosorpti on. The firs t and foremost reason bei ng, biosorption basicall y involves an exchange of two metals . Thus, the overall reac ti on can be e ither endo or exothermi c. The energy liberated due to the binding of o ne ion is compensated by absorption of the same by the io n re leased. Second ly, apart from ion-exchange the phenomenon in volves complex fo rmation as well. As biosorption

occurs in a ve ry narrow temperature range (5° to 40°C) temperature e ffect s are o nly of secondary importance.

Gold sorptio n by Azolla fiheuloides was found to be independent of temperature within the range 1O_50°C45

. Suhasini el al85 observed a decrease in Co sorption with inc rease in temperature fro m 190 mg/g at 30°C to 168 mg/g at 45°C by the biosorbent PFB I . Sim il ar reports of decrease in Ag sorptio n by

Saccharomyces eerevisiae from 185 /-1mo l Ag/g dry

weight at 4°C to 168.4 /-1mol Ag/g dry weight at 55°C has been reported by Sing leton and Simmons84

.

Puranik and Paknikar38 have observed no significant difference in specific metal uptake by Cilrobacler biomass in the temperature range 4° to 55°C. Optimum silver sorption by micromyce te was

observed in the temperature range 30° to 50°CY1•

Upon a decrease in temperature to 6°C or an increase to 80°C the level of s il ver ex tracti on reduced by 10-20%. Bai and Abraham88 have reported higher Cr removal efficiency by Rhizopus nigrieans biomass at

GUPTA & MOHAPATRA : MICROBIAL BIOMASS FOR REMOVAL OF HEAVY METALS 955

temperature above 30°C; nevertheless there was a decrease in sorption at higher temperature of 50°C. They attributed this decrease to the possible damage caused to the active binding sites in the biomass.

Pre-treatments As biosorption process involves mainly cell surface

sequestrati on, cell wall modification can greatly alter the binding of metal ions. A number of methods have been employed for cell wall modificat ion of microbi al cells in order to enhance the metal binding capacity of biomass and to elucidate the mechanism of biosorption . These modifications can be introduced at two stages: either during growth or to the pre-grown biomass.

During the growth of microorgani sm the condition in which a microorganism is grown can determine the biosorption potential since it affects the cell surface phenotype96 Many reports exist relating to the effect of culture conditions of cells on the biosorpti ve

. f d f ·97 '18 H h . capacity o ' yeast an ungl · . owever, not muc IS known about the changes in biosorptive capacity of algal biomass and cyanobacterium. In Phormidium laminosum, biosorption decreased initi ally with nitrogen starvation and subsequently increased until it reached the value of nitrogen sufficient cellslJ9

In the pre-grown biomass several physical and chemical treatments have been tried to tailor the metal binding properti es of biomass to specific requirements JOo. The physical treatments include heating/boiling; freezing/thawing , drying and lyophili zation while the chemical treatments invol ve the use of alkali , acid, organic solvents and salt solutionsl<l l. Sar el al. 35 have reported enhancement of both Ni and Cu sorption by alkali treatment (NaO H 0.1 mM) in case of Pseudomonas aerug inosa. Acid treatment with HCI (0. 1 mM) did not stimulate sorption whereas autoclaving and oven heating sli ghtly inhibited the sorption. Among organic chemical s tested toluene and ethanol slightl y enhanced sorption in contrast to acetone which inhibited metal sorption . Methanol- chloroform mixture did not alter metal sorption whereas lysozy me pretreated biomass showed reduced sorption of both the metal s. Most severe decline in metal sorption was observed following pretreatment with SDS. Puranik and Paknikar38 have reported 13-68% decrease in Pb sorption due to heat and chemical treatment and 22-78% decrease in Zn sorption after most other pretreatments in case of Cilrobacler strain MCM 8-1 8 l. In contrast to these, pretreatment of the biomass with

NaOH, Triton X-IOO, Na2C03, (NH4h S04, ethanol, methanol and acetone resulted in 37-109% increase in Cd uptake. Treatment of Saccharomyces cerevisiae biomass with NaOH or SDS at lOO°C for 30 min decreased the Ag biosorpti on capacity of the biomass to 43 .5% and 70.9% respectivell 4

. However, no other treatments (HN03, (N H4h S04 or EDT A) has any effect on Ag biosorption. Na2C03 and ethanol followed thi s while H2S04 had an adverse effect on biosorption . Pre- treatment of Sargassum biomass with Ca, Na, Mg and KOH revealed that KOH washing resulted in stable biosorbent with improved affinity for Zn 102. The stability of the biosorbent following KOH treatment can be attributed to less loss of organic carbon due to the treatment while ac id treatment caused much damage to the biosorbent and resulted in greater loss in total organic carbon. Streptomyces rilllosus biomass pre-treated with NaOH (1 mol/dm3) was able to bind more Zn than the untreated one lO3 . Baik et al.79 observed that pretreatment of Aspergillus niger, Rhizopus oryzae and Mucor rouxii with NaOH (4M) at 121 °C enhanced drastically the sorption of Cu, Cd , Ni and Zn as co mpared with the untreated biomass. Fourest el 01. 104

have reported cationic activation of metal sorption following saturation of Rhizopus arrh izus, Muco r miehei and Penicilliwn chrysogenum biomasses with Ca.

Mechanism of biosorption Sound understanding of the chemical nature of the

metal ions and the binding sites is imperati ve and indi spensable to develop an understanding towards the mechani sm .of biosorption. On the other hand, compl ex nature of the biopolymers poses a hindrance in understanding the mechani sm of the biosorption. Preliminary studies carried out on biosorpti on reveals it to be a complex interplay of the propert ies of the biomolecules of the cell wall and the chemical nature of the metal ion in question . Each of the microbial group is characterized by a di stinct cell wall structure. Biosorption by these microbes is attributed mainly to the li gands present in the biomolecules of their ce ll wall polymers. Hunt l05 has elucidated the role of various biopolymers in metal biosorption.

Binding sites The cell wall polymers provide a multitude of

chemical groups such as hydroxyl, carbonyl , carboxyl, sulfhydryl, thioether, sulfonate, amine, Imine, amide, imidazole, phosphonate and

956 INDI AN J EXP BIOl, SEPTEMB ER 2003

phosphodiesterl 5. These chemical groups of the

biopolymers in turn harbor the binding sites, which provide the ligand atoms to fo rm comp lexes with

I · 105 106 T bl 3 I ' . I b' d ' meta Ions . . a e Ists varIOUS meta 111 ll1g groups in bi ological polymers. Each of the binding sites can participate in different binding mechani sms such as complexation and electrostati c attraction of metal cations. Consequently several mechani sms act in combinati on. In general for metal binding we can distingui sh between ion exchange, sorption of electrica lly neutral material (soluble metal ligand complexes) to specific binding sites and microprecipitation. These main mechani sms are based on sorbate-solvent interactions, which in turn rely on some combi nation of covalent , electrostatic, and Van del' Waal's forces.

Importance of the given group for biosorption of certain metal by certain biomass depends on various fac tors such as (i) quantity of sites in the biosorbent materi al (ii ) chemical state of the site (i.e. its ava il ab ility) (iii ) access ibility of the site (iv) affinity between the site and the metal (i.e. binding strength ). For covalent metal binding even an already occ upied site is theoretica ll y ava ilable. But to what extent thi s site can be used by the metal ion for binding depends on the bi nding strength and concentration of the metal in co mparison to that already occupying the site. For electrostatic binding a site is ava il able onl y if it is ioni zed 15 .

Metal affinity towards biomolecules The affinity of various metal vari es with respect to

the bimolecular ligand. The bond character in biosorpti on can be explained parti all y by Pearson's concept of hard and soft metallic ions lo7 . Thi s scale is based on the binding strength of the ions with F and

r. Those metallic ions (e.g. Na, Mg, K, Ca etc.) which form strong binding with F- are referred to as 'hard ' while those forming relatively weaker bonds (e.g. Au, Ag, Pb, Hg etc.) are referred to as 'soft' ions. A very and Tobin lo8 have studied the applicabi li ty of ' hard and soft ' principle in predicting metal sorption by Saccharomyces cerevisiae. There also exists a class of ions with intermedi ate degree of hardness (e.g. Zn, Cu, CO, Ni, Fe etc.) and are refen'ed to as ' transition' metals. The hard ions also serve, as essenti al biologica l nutrients whil e the soft ions are usually tox ic in nature, The transition metals of intermedi ate hardness are less toxic and are present in certain biomolec ules where they ass ist in mediating spec ifi c biochemical reactions 109 . Among the ligand atoms '0 ' and 'F' are sonsidered hard, 'S and 'P' are considered soft while 'N' stays in the intermedi ate category .

The hard ions in biological system fo rm stable bonds with hydroxy l, phosphonate, phosphate, carboxy l and carbony l group ; all of which contain '0 ' atoms. The soft ions on the other hand from very strong bonds with sulfhydryl, amine, imidazole, amide and imine groups i.e. groups rich in Sand N atoms. The hard ions main ly demonstrate ionic binding whi le the soft ion displays a covalent character l09,

Nieboer and Richm'dson 110 have classified the metal ions into class A (0 seeking), ' class B (N/S seeking) and class C (boderline or intermedi ate character), based on their binding preferences towards the 0 , N or S containing ligands of biomolecul es,

Overall mechanism of biosorption As mentioned earlier the phenomenon of biosorption

may inc lude a combination of several mechanisms such as electrostatic attraction, complexation,

Tabl e 3 - Metal binding groups in bio logical poly mers

Chemi cal group

Hydroxy l Carbony l (ketone) Carboxy l

Su lfhydryl (thiol )

Thi oether Su lfona te Amine

Secondary amine Imine A mide

Imidazole Phosphonate

Phosphodieste r

Structu ra l form ula

-OH

>C=O -COOH - SH

>S

-N H2 >N H = NH -CON H2

-P-2(OH)=O >POOH

Occurrence in biomolecu les ligand atoms

Polysaccharides, uroni c aci d and amino ac ids 0 Pe ptides and prote in s 0 Uronic ac ids, amino ac ids 0 Amino ac ids S Amino ac ids S Sulfated polysaccharides 0 C hi tosan, am ino acids N

C hitin , peptidog lycan, peptide bonds N Amino acids N A mino acids N

Amino ac ids N Phospholipids 0 Teicho ic ac id, lipopolysaccharide 0

+

GUPTA & MOHAPATRA: MfCROBIAL BIOMASS FOR REMOVAL OF HEAVY METALS 957

ion-exchange, covalent binding, Van der Waal's f d . d· .. . III 11 3 orces, a sorption an mlcropreclpltatlOn . .

Complex formations in general involve both covalent and electrostatic components, whose relative contribution can be estimated by knowing how specific the binding is. In case of electrostatic attraction the binding strength correlates directly with the charge density (Z2/rhyd). This implies that the ions of the same charge (z) and hydrated radius (rhyd) should be bound with equal strength. A major deviation from this ~inding strength indicates a tendency towards covalent bond character l5. Knowledge about the ions re leased provides information about the bond type. Yang and Volesk/ I have reported biosorption of Cd to be accompanied by the release of hydrogen protons from protonated non-living biomass of Sargassum jluilans . They observed that the uptake of Cd and release of protons matched throughout the biosorption process. Singleton and Simmons84

observed increased release of H+, Mg2+ and ci+ with

a corresponding increase in Ag biosorption by Saccharomyces cerevisiae . In general electrostatically bound ions cannot di splace covalently bound ions. As observed in certain cases proton release occurred only during heavy metal uptake and not during light meta l uptake. As protons are bound covalently, these heavy metals must have bound more covalently than that the light metal ions.

Similarly inhibition of metal sorption by Na, Ca or Mg implies greater electrostatic binding of these ions in comparison to other metal ions l1 4. As mentioned earlier concomitant release of Ca and Mg ions during Ag sorption by Saccharomyces cerevisiae has been observed84. Reports of release of monovalent and divalent ions during biosorption has also confirmed by instrumental analysis82.115 .

Apart from the above mentioned mechanisms there exists the phenomenon of ' adsorption' and ' microprecipitation ' which describes the accumulation of electrically neutral metal ions without the release of any other bound ion27. The phenomenon of adsorption is driven by the affinity between the sorbate and the sorbent while in case of microprecipitation it is driven by the limited solubility of the solute in the solvent. A less hydrophilic molecule has lower affinity for the liquid phase and consequently gets adsorbed more easil/5. Microprecipitation of the metal cations and anions which are often the metabolic products of certain biomass types form insoluble aggregates (as salts or complexes) such as sulfides, carbonates, oxides, oxalates and phosphonates82. 11 6-11 8. Transmission

electron microscope studies carried out on Rhizopus oligosporus revealed the deposition of Pb only on the surface of cell wa1l82 . Figueira el al. 11 8 have rep0l1ed the complexation of Fe2+ and Fe3+ with sulfate and carboxyl groups of Sargassum biomass. Swift and Forciniti 11 7 observed the deposition of lead as lead phosphate prec ipitate in the vicinity of the cell wall of Anabaena cylindrica. The pH of the system also goes a long way in influencing the process of

. . .. 58 11 9 G I PO h d mlcropreclpltatlon . . reene et a . - ave reporte interference of HC03- in a pH dependent manner during U sorption to Chlorella vulgaris.

During ion-exchange mechanism of metal sorption the charge of the ions taken up by the biosorbent equals the charge of the ions released. This results in maintaining the neutral charge of the biosorbent. The acquisition of these charged ions in turn can occur through a variety of other mechanisms such as electrostatic attraction or complexation . Regardless of the mode, the main driving force behind the mechanism is the attraction of the metal ion for the biosorbent.

Discerning the groups involved in biosorptioll One of the major challenges in knowing the

chemical groups involved in biosorption is the complex nature of the microbial biosorbent material.

Techniques such as modification/blocking of chemical groups have been used for indirectly deducing the mechanism of biosorption . Ashkenazy et al. 121 investigated Pb biosorption of acetone washed yeast biomass by chemical modifications of the cell wall components. Propylamine was used for parti al as well as complete acetylation of hydroxyl and am ino groups. Also, amino group modifications were carried out by reacting these groups with tri-nitrobenzene sulfonicacid (TNBS) and aminosuccinylation reaction. The authors observed an increase in biosorption following modification of amino groups while acetylation of the hydroxyl group decreased the biosorptive capacity. This indicated involvement of negatively charged carboxyl group and amino group in Pb biosorption. Carboxyl groups were suggested to be involved in binding Cu and Al in algal species, as blocking of carboxyl groups by esterification lead to a decrease in metal binding l22 . In Sargassum nalans, the functional groups, viz. carbonyl (C=O) and amine (-NH2) were found to provide binding sites for metals 123.124. Akhtar el al. 125observed 90% decrease in

metal sorption by fungal biomass following chemical modification of carboxylic acid functional groups.


The advent of modern instrumental techniques like electron microscopy, energy di spersive X-ray analysis (ED AX), infrared spectroscopy (IR and FTIR) and electron spin resonance spectroscopy (ES R) has helped in developing a better understanding regarding the in volvement of the functional groups in the bond fo rmation process. Table 4 li sts the analys is carried out to reveal the groups/ligands in vo lved In

biosorption. Electron microscopic techniques such as SEM and TEM are helpful in carrying out the studi es on locali zat ion of the metals caused due to precipitation , formation of intracellular complexes. TEM of Rhizopus oligosporus cell s exposed to Pb revealed the deposition of Pb only on the surface of cell wa ll s82 .

X-ray spectroscopic techniques are based on the principle that a matter can absorb X-rays, giving ri se to X -ray absorption spectra. The fundamental frequencies observed are charac teri stic of the functional groups concerned and are absolutely specific. This gives ri se to the term fingerprint for infrared pattern obtained. As -the nllInber of functional group increase in more compl ex molecu les, the absorption bands in the IR pattern become more difficult to ass ign. However, in such cases group frequencies ari se that help to simpli fy interpretati on. These groups of certain bands regu larly appear near the same wavelength and may be ass igned to specific molec ular groupings. Such group frequencies are extremely valuab le in structural diagnosis . The freq uency associated with a particul ar group varies sli ghtl y, owing to the influence of the molecular environment. Such vari ati ons are extremely useful in structural biochemi stry studi es where they he lp to di stingui sh between bond vibrat ions of different chemi cal groups. For example the C-H bond variation in methylene (-CH2) and methyl (-CH3) group, C=O vari ation in carboxy l (-COO H) and carbonyl (>C=O) group . A decrease in wavelength also occurs when double bonds are formed as the stretching frequency increases. FTIR technique has been widely used for discerning the functiona l groups in -

I d · b' . 58 12) 124. 127 vo ve In losorptlon . . . The energy di spersive X-ray spectroscopy analys is

is based on the princip le that X-rays can be absorbed by matter, which gives ri se to X-ray absorption spectra . These X-ray di spersion spectra may be detected at various angles that can then be co-related with the complex formed. EDAX analys is of Pseudomonas aerug inosa ex posed to Cu revealed it to be accumulated principally as CuS, Cu)N, Cu)p and CuO I26.

The electron spin resonance spectroscopy (ESR) employs the magnetic phenomena of the charged particles. This magneti c phenomenon in molecu les ari ses due to the spin of the charged parti cles i.e. electron s. Energy is requi red to cause thi s resonance condition, which is absorbed and recorded as peak in the ESR spectrum. Th is indicates the presence of paramagneti c species. The area under the peak is proportional to the concentration of the species. Calibration of the ins trument with known standards all ows the concentration to be calculated. Philip e l

al.11 5 usin g thi s technique have reported the '0' group of carboxy l peptidoglycan and ' N' of am inosugars or structural proteins to be assoc iated with Cu binding on Bacillus polymyxa .

X-ray photoelectron spectroscopy fo r chemical analys is (ESCA) or simply X-ray photoelectron spectroscopy (X PS) is a relatively new technique fo r determ inati on of binding energy of electrons in atoms/molecules, whi ch depends on di stribution of valence charge and thus gives inforlllation about the ox idat ion state of the atom/ion. Pethkar et al. 127 using thi s technique have reported the in volvement of' , ato m in Ag sorption by Cladosporium cladosporioides sp.2 at higher pH .

Mathematical considerations in biosorption studies

Application of adsorption isotherms in biosorption studies

The biosorptive metal uptake of an organism can be quantitati vely evaluated fro m ex perimental biosorption equi librium isotherms similar to those used for the performance evaluat ion of acti vated carbons. The graphical expression is a plot of the specifi c metal uptake (q) by the biosorbent against the residual metal concentration. The resulting plot is of len hyperbolic with the uptake va lue often reaching a sta tionary phase as it approach's saturati on at hi gh concentrati ons of the adsorbate. The two widely accepted and linearized adsorption isotherm models used in the literature were those proposed by Langmuir l29 and Freund lich 130 and being named after them. The general form of the Langmuir model equation is

q =qobC/l + bC where q specifi c uptake at residual/equilibrium

qo C

and b

concentrati on max imum uptake residuai/equi librium concentrati on constant rel ated to energy of adsorption

t

GUPTA & MOHAPATRA : MICROB IAL BIOMASS FOR REMOVAL OF HEAVY METALS 959

Table 4 - U~e of instrumental analysis for determining/predi c ting chemi cal groups involved

Organism Analysis Bonds/Groups involved References

Pseudomonas aerugillosa

Ganodenna lucidium

Bacillus polYlllyxa

Anabaena cylilldrica

Rhizopus o/igosporus

(EDAX )

EPR

EPR

EDAX coupled with SEM and TEM

TEM and EDAX

Aspergillus niger. Penicillium FTIR chrysogenu11l, Mucor miehei

Chlorella vulgaris

Sargassum IWlans

Pseudomonas ael'llginosa

XANES (X- ray Absorption Near Edge Structure)

EXAFS (Extended X-ray Absorption Fine Structure)

ESC A or XPS and FTIR

EDAX

Rhizopus spp. Based biosorbent EDAX and EPR

Rhizoplts oligosporus T EM and EDAX

Cladosporium cladosporioides XPS and FTIR

Pseudomonas aerugillosa IR and EDAX

Chi orella fusca X-ray spectroscopy

Cu accumu lated as CuS. Cu)N, Cu)p and CuO 126

'0' dominating atom in binding 31

'0' of carboxyl group of peptidoglycan and 'N ' of II S aminosugars o r struc tu ra l prote ins to be associated with Cli binding

Pb deposited as polyphosphate bodies 11 7

T EM studi es revealed deposition of Pb onl y on the su rface 82 of cell wall. EDAX revea led disappearance of peaks corresponding to Mg2+, S2+, K+ and appearance of Pb2+ along with existing p+

Uranyl binds to amine and amide sites . UO vibration band appears at a wave number which varies according to pH and nature of the metal ion spec ies in solution

Au was bound in + I ox idati on sta te i.e. reduction occurred from Au3+ to Au 1+. Under hi gh concentration of AlICI), red uct ion was parti al.

AuCI ) is bound in co-ordination with 'N or '0' ato ms (EXAFS cannot distinguish be tween nearest ne ighbors in a periodic table). In AuCI 2 the principle binding atom was'S '

ESCA revealed the deposition of gold in e lemental form (AuO) whi le FTIR indicated the involve me nt of carbony l (>C=O) and amino groups in the metal binding.

Lanthanum uptake by d isp lacement of Ca & Mg

EDAX revealed replacement of Ca ions by Co while EPR showed the involvement of free organic rad ical (not specified)

TEM revealed e lec tron dense a rea on the middle sec tion of the cell wal l following ex posure to Cd. EDAX spectrum conspicuous P and Cd peak indi cating precipitation of insoluble metal lic Cd as P04-

3 precipitate

Strain 1: XPS revea led that at low pH 2.0, 'C' and '0' group not involved in gold binding.

Binding of Au was accompanied by appearance of 'N- . Higher pH 7.0 showed presence of 'C', '0' , ' N' ~ II of which participated in Ag bi osorpt ion.

Strain 2: Gold inte racts with ' 0 ' and ' N' peak while Ag reacted with the 'N' g roup only.

Strain 1: FTIR analys is revealed unmaski ng >C=O and -COOH group which did no t change even afte r exposure to Au , implying binding of Au as AUCI"4 anion at pH 2.0. In volvement of CoO and C=O in Ag sorption.

Strain 2: CoN and CoO were probably involved in AU binding . C-H, N-H_ O-H, CoO and NH3+ a ll were in volved in Ag sorption.

X-ray diffraction revealed the presence of Cu as CuO as well as CuS while Ni was present as P, Nand C.

FTIR revea led the precipitation of carboxy l, carbony l and phophoryl groups along with H-bonding in metal so rption.

Cu sequestered as polyphosphate bodies

S8

20

123

36

8S

69

127

3S

128


Taking the inverse of both sides, the equation can be linearized as follows:

IIq = I/Q l11ox .b.C + IIQl11ax

The Freundlich adsorption model has the general form

q=k C l /n

This can be lineari zed by taking the natural logarithm of both sides of the equation to give,

In q=ln k+ lin In C

The intercept In k gives the measure of adsorbent capacity while the slope lin gives the intensity of adsorption.

The Freundlich isotherm is more preferred for aqueous solutions whi le · the Langmuir isotherm is preferred more for gaseous solutions. Though both the models describe biosorption data well, but they are not models whose terms and parameters would have a convenient and appropriate physical interpretation attached to them I. An isotherm that is steep at the orig in at low residual concentrations of the sorbate is highl y desirable because it indicates a high affi nity of the sorbent for the given sorbed species. Such a biosorbent would be performing well at very low concentrations of the sorbed species in the solution .

Blanco el al. 72 have reported high Langmuir constant (Ql11ax) values of 19.53, 17 .S3, 16.10, IS.05 mglg for Cu, Fe, Ni and Zn respectively for polysulfone immobili zed Phorl1lidiul1L laminosum while that of the epoxy immobili zed beads were 2 1.2S, 16.7, 13. 13 and IS.60 for the respecti ve metals. The binding energy values ranged between 0.06 for Ni to 0.36 for Cu in case of polysulfone immobili zed beads and between 0.10-0.26 for epoxy immobili zed beads. Sar el al. 35 have evaluated the Ni and Cu sorpti on capacities of lyophili zed Pseudomonas aem ginosa cell s using Freundlich isotherm. They observed that the sorption intensity In k for Ni (0.69) was higher than that of Cu (0.52) while the sorption in tensity ( lin) of Ni (3. 14) was lower than Cu (4.2), implying that in Ni biosorption equilibrium metal concentration pl ays an important role as compared to that in Cu. They also observed that at lower equilibrium concentration (Ce 20 mg/L ), Ni and Cu removal capacity was almost the same whi le at higher concentration (Ce 200 mg/L) , N i (121.5 mg/g ) was preferred over Cu (66 mg/g ).

Rai el al. 131 have compared the biotechnological potenti al of laboratory grown and naturally occurring Microcyslis for Ni and Cd biosorption using Freundlich, Langmuir and BET isotherms. Freundlich and Langmuir constants revealed higher affinity for

Cd than for Ni. High R2 values of BET isotherms suggested a mul tiplayer binding of metals. Puranik and Paknikar38 have obtained high co-relation coefficients for Pb, Cd and Zn biosorpti on (0.99, 0.99 and 0.95, respectively) by Citrobacter strain MCM BlSI using Langmuir isotherm. Highest Qmox value of 5S .78 mg/g biomass with b of 0.11 was obtained in case of Pb while lowest QIll"X = 23.62 mg/g and b of 0.16 was obtained in case of Zn . Sar and D' Souza37

obtained a good fit of lineari zed Langmuir and Freundlich adsorption isotherms for live as well as lyophilized biomass of Pseudomollas . Philip el cd. J6observed that U sorption by Pseudomonas aeruginosa fo llowed Langmuir and Freundlich isotherms but not BET isotherms. Zhou 93 compared the Zn adsorptive capacity of six di fferent fungi (Rhizopus arrhizus, Mucor racemosus. Mycotypha africana, Aspergillus nidulans. Aspergillus niger and Schizosaccharomyces pombe) by Freund lich and Langmuir isotherm mode ls. Amongst the fungi tested R. arrhizus had highest K and Qlllax va lue of 11.055)J.mol l

.n In/g and 213J.lmo]Jg, respec ti vely.

Korenevskii et al.9l evaluated the biosorpti on efficienci es of micromycete cultures u ing Freundlich and Langmuir constants. They obtained low K, va lue, which were indicative of hi gh affinity of the fungal biomasses for Ag cation s. Two to three fo ld variations in Ag sorption capacities was observed among all the micromycete species. Further vari ations were not only subjective to species but also to strains of the same spec ies91

. Bai and Abraham88 observed th at eq uilibrium data of Cr sorption by Rhizopus nigricans fitted well into lineari zed Freundlich isotherm mode ls. Say et al.24 have reported good fit of Langmuir model to Cd, Pb and Cu sorption by filamentous fungus Phaenerochaete chrysosporium . Wong et al.132 have compared the Ni sorption capac ities of two Chlorella spec ies - C vulgaris and C miniata. They have reported max imum Ni uptake to be 641.76 and 1367.62)J.g/g biomass for C vulgaris and C minia/a, respectively. The experimental values followed the same trend as predicted by the Langmuir adsorption isotherm model (29S5.07 and 12S2.05 )J.g/g biomass for WWl and C vulgaris respectively) . Further high correlation co-efficient for the regress ion lines indicated good fit of the model to both the systems. Lower n value of C miniata ( 1.33) compared with C vulgaris (1.5 I) also indicated that the adsorption by the former was more effec ti ve at low metal ion concentration 132 . Cd biosorption by Ca-alginate immobilized Trametes versicolor fitted both into the

GUPTA & MOHAPATRA: MICROBIAL BIOMASS FOR REMOVAL OF HEAVY METALS 96 1

Langmuir and Freundlich adsorption isotherm mode ls with very high R2 val ues23 .

Modeling biosorption process with special emphasis to multimetat situations

The application of mathematical models to biosorption processes apart from giv ing a quantitati ve description of the process also aid in optimi zing the operating conditions. Secondly , mathemati cal modeling at bench scale levels he lp in minimi zing the number of ex perimental runs and he lp to pin-po int the results of spot check ex periments IS. Thirdly, modeling at industriai scale becomes indi spensab le as it goes a long way in reactor des igning and he lp discover bottlenecks thereby optimi zing the operational conditions and reduc ing the costs in vo lved in hit and trial runs.

Though both the Freundlich and Langmuir isotherm models predict the metal uptake as a fun ct ion of the concentration of one metal i.e. in the ir basic fo rm they are suited fo r mo no meta l systems without pH effects. Thus, they are not appropriate for ionexchange systems, whi ch usua ll y in vo lve mo re than one metal spec ies. Chong and Volesky l33 have described a mUlti-component Langmuir isotherm model, which assumes a 1: I stochio metry between metal ions and binding sites whereby all metals make use of the same sites and compete for them. The authors have used three dimensional sorption isotherm surfaces usi ng the software MATLAB 4.0 to evaluate the twometal sorpti on performance of the biosorbent. In the ir studies on modeling of proton-meta l ion exchange in biosorption , Schiewer and Volesk/ 34 have extended the Langmuir model for predictions of competition between divalent heavy metal ions ( 1:2 stochio metry ). They have rendered direct calculation of meta l uptake without the invo lvement of any iterati ve calcul ations. Schiewer and Voleskyl s have discussed models invo lving competition among metal cations, pH effects, and effects of io nic strength and e lec trostatic att racti ons. Further, they have also di scussed in detail quantitative modeling of batch kinetics. Chong and Volesky 135 have evaluated the extended mUlti-compo nent Langmuir model for ternary metal solutio ns of Cu, Cd and Zn. They reached the conclusio n that fitting of the multi-component Langmuir data to the tern ary data was semi-empirical and some predictions of the behav ior of ternary meta l systems were in a reasonably good agreement w ith the experimental results and those derived from binary systems. The use of tri angular diagram technique was successfully · imple-

men ted for the graphic representation o f ternary bio-. d 13S sorption ata .

Sanchez el al.62 investi gated three models to propose the most suitab le equation to represent the sorption data of Cu-Zn system in 3D space. The first model produced an eq uatio n with three parameters, the second and third had fo ur and five parameters, respec ti vely. These parameters were eva luated using MATLAB 4.0 program. The in vesti gations revea led that a ll the three models studi ed could make a good prediction of the metal uptake for the system studied with minimum variance. Thus, the cho ice of the best model was restricted by looking for one with the lowest number of parameters i.e. model 1 which they used for the constructi on of 3D biosorption isotherms. This model was a binary Langmuir type equation.

Sag el al. 136 had a lso employed the compet iti ve Freundlich and Langmuir adsorpt ion models to study the si multaneous biosorption o f Cr and Fe on C. vulgaris and R. arrhizus. They reported the Freundli ch model for binary meta l mi xtures to be sat isfactory for mos t adsorption eq uilibrium data of Cr and Fe io ns on C. vulgaris, whil e the competitive and modified Lang muir models were more suited to characteri ze competiti ve adsorptio n of C r and Fe from binary systems by R. arrhizus. Sag el 0 1. 137 have analyzed the eq uilibrium data of Pb, C u and Zn sorption from binary and ternary metal so luti ons using empirical competiti ve Freundlich isotherm model. Thi s isotherm model is related to the indi vidual isotherm parameters and takes into account the correction factors.

Mehta and Gaur l38 have reported the use of twodimensional contour plots using the graphical software Sigma Plot 2.0 to depict the conCUITent sorption of Ni and C u by ChIarella vulgaris. Thi s represented a re latively simpler approach where the ex te rnal concentration of meta ls were plotted on the X and Y ax is while the contour lines corresponding to Z-axis represented the corresponding metal sorbed. Swift and Forciniti 11 7 have developed a mass-transfer kineti c model, whi ch quantitati ve ly predicted the concentration of Pb in ce ll s of Anabaena cylindrica as a function of spatia l dimensions and time. This model deals with on ly a mo no meta l situation .

Recent advancement in software technologies has also come a lo ng way in developing software that is spec ific for predicting biosorption performance in fixed bed reactors. Figueira el al. 102 have tested one such software package - ' IMPACT' , for evaluating the biosorpti o n performance of Sargassum biosorbent


with a metal mixture (Cu, Cd and Zn) in a column. They have reported that the application of experimental IMPACT computer software was only partially successful in exactly simulating the biosorption column performance.

Elution and recovery of metal There are generally two fates of the metal laden

biomass; either it is 'ashed' off' (by incineration) or regenerated (by elution). The first alternative is preferred where the biosorbent material is supplied as a waste biomass - cheap and abundant. This reduces the volume of waste generated. The more desirable and economic option of regeneration is based on the selective stripping 'off' of the metal laden on the biomass by the use of desorbing agents . The desorbing agents or eluting solutions function by uncoupling the bonds formed between the metal and the biosorbent. Small quantity of the eluant results in high metal concentration in the resulting solution , thus making it amenable to easy and economical extraction procedures. Thus the solid/liquid ratio (S/L ) is often used to express the efficiency of the eluant. The solid represents the amount of the biosorbent and the liquid represents the volume of the eluant applied. High SIL values are desirable for complete elution 1.22.

Chelating agents, sa lts and alkali solutions proved to be the best eluants22 .38.4o.61 .62.7o.91 while mineral

ac ids though elute considerable amount of metal22.35.39.85. 139, ususally cause damage to the

biosorbent. This in turn affects the 'second resorption ' and subsequent resorption cycle61 . Philip et al. 36 have reported 95% desorption of the loaded U from Pseudomonas aeruginosa using 0.2 M HC!. They however observed an adverse effect of the acid on the viability of the cells. This drawback resulted in opting for citrate buffer (0.2 M, pH 4 .0) as the eluant of choice with 80% desorption. Similar decrease in biosorption capac ity of beads has been reported for elution of metal laden beads of Citrobacter biomass by 0.1 M HC1 38. Addour et al .103 have reported that Streptomyces rimosus biomass regeneration with 0.1 mol/dm3 HCI resulted in 90% Zn recovery with 20% weight loss.

However, reports do exists where the use of acid eluants did not affect the biosorptive potential of the biosorbent in subsequent cyc]es35.60.64.86.87 . Kama et al.60 have reported 0.1 M HCI to be effecti ve in eluting Cd and Co. They observed 80% Cd binding ability to be retained up to three cycles while in case

of Co the loss of the binding ability was rapid with more than 70% of the initial binding capacity being lost. Suhasini et al.85 have reported relatively high readsorption efficiencies (>70%) for PFB 1 following desorption with HC] or H2S04 (0.1 N). Arica et al.23

observed no noticeable change in the adsorption capacities for Cd by Trametes versicolor following desorption of the metal laden biomass with HCI (10 mM). Yan and Viraraghavan73 have reported comparable readsorption capacities of polysulfone immobilized Mucor rouxii biomass following desorption with 0 .05 N HNOJ . Huang and Huang78

have enhanced Cu sorption by ac id washed Aspergillus oryzae while acid washing did not adversely affect the metal adsorption capacity of Rhizopus oryzae mycelia. They have thus recommended acid wash for the dual purpose of biomass pre-treatment as well as regeneration .

An important aspect of the metal elution is the selective and consecutive removal of metals from the biosorbent. Ahuja et al.53

-55 observed maximum

elution of Zn , Cu and Co by EDT A (10 mM), citrate buffer (0.2 M, pH 3.0) and Na2C03 (1 mM), respectively . Darnall et al. 140 have reported a scheme for selective recovery of Cu2+, Zn2+, Au3+ and Hg2+ bound to polyacrylamide immobi lized Chlorella vulgaris by lowering the pH from 6.0 to 2.0 and subsequently treating the column mercaptoethanol to elute Au3+ and Hg3+.

Ideally speaking several criteria have been laid down for the choice of desorbing agents. These include high SIL rati o, high metal concentration factor (after desorption and before adsorption), high efficiency, fast kinetics, selectivity, preservation of structural integrity of the biosorbent , economical and environment friend ly. However, due to selfcontradicting requirements it often becomes difficult to look for an eluant possessing all these major criteria.

Commercial biosorbents Various types of microbial biomasses have formed

the basis of formulation of new and potent metal sequestering biosorbents. This is important in the present day scenario, as there is an increas ing need for an effective and economical process to remove metal ions from industrial wastewater and drinking water.

A potent algal biosorbent AlgaSORB ™ has been developed using a fresh water alga Chlorella vulgaris to treat wastewater l40

. Thi s can efficientl y remove metallic ions from dilute solutions i.e. 1-100 mg/L

GUPTA & MOHAPATRA : MICROBIAL BIOMASS FOR REMOVAL OF HEAV Y METALS 963

and reduces the concentration of metals down to 1 mg/L or even below. Calcium and magnesium also does not affect the sorption of heavy meta ls by AlgaSORBTM.

Another metal sorption agent AMT-BIOCLAIMTM (MRA) has employed Bacillus biomass to manufacture granulated materi al for waste water treatment and meta l recovery . This can accumulate metal cations with efficient removal (>99%) from dilute solutions. It is non-selective and metal s can be stripped from it after loading by H2S04, NaOH or complexing agents and the granules can be

c ?6 regenerated lor repeated use- . Bio-Fi x biosorbent uses biomass from a variety of

sources including cyanobacterium (Spiruiina), yeast , algae, plants (Lemna sp.) and guar gums to give a consistent product and immobili zed as beads using polysulfone. Zinc binding to thi s biosorbent is approximate ly 4-fold higher than the ion-exchange res ins. There is vari abl e affinity for different meta ls Al > Cd > Zn > Mn and a much lower affinity for M g and Ca. Metals can be e luted using HCI or HN03 and biosorbent can be reused for more th an 120 ex traction-e lution cycles.

T wo marine a lgae namely, Sargassum natans and Ascophyllum nodosum have been found to have excellent biosorption capacities for gold and cobalt respectively , among the photoautotrophs 11.141 .

Conclusions Biosorpti on IS an economically feasible;

technically efficient technology for metal removal/recovery and can comfortably fit into the metal treatment processes and is eco-friendly in nature. Inspite, of these advantages why has the biosorption/wastewater treatment remained as an embryonic industry ? This is because not all the companies, which generate metal polluted wastewater, will have the capability or the interest to do anything other than the basic treatment to comply with the legislati ve. Hence to overcome thi s what is needed is a series of specialists, centralized facilities which would be capable of re mov ing meta l from waste water and regenerating or process ing the metal loaded sorbent and then converting the recovered metal into reusable form. Alternatively, if the biosorbent used is a waste product, its inc ineration could be used to produce meta l rich slag.

Looking into the economjcs, feasibility in te rms of scale up and working efficiency as a technology the microbial bi osorbent prov ide encouraging results to

be utilized in wastewater treatment. What is needed is an extra moral input from the industries generating metal polluted wastewater to in vest into such clean up technologies before di scharging their liquid effluents into the water bodies.

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