Immobilization of Yeast and Algal Cells

for Bioremediation of Heavy Metals

John R. Duncan, Dean Brady, and Brendan Wilhelmi
1. Introduction
The technique of selective broaccumulation of heavy metals by microbral
systems offers a possrble approach for the remediation of contaminated water
bodies. Although heavy metal bioaccumulation is a well known and understood
process and has been used on a small scale wrth some success,problems with
containment and reactor design have rendered large-scale engineering of broaccumulation systemsimpractical.
As a meansof nnmobilizatron of the biomass,gel rmmobilizatron (see Chapters5
and 6) has been used with some success.Sacchuromycescerevzsiae has prevtously
been unmobrl~zedin polyacrylamide and used for broaccumulatton of Cu*+, Cd*+,
and Co2+(I), whereasimmobilization of the fungus Rhizopus arrhizus has beenparttcularly successfulm the accumulatron of Cd*+, Cu*+, Fe3+,Mn+, Pb*+,and Zn*+,
and in the recovery of uramum from ore bioleach (2-4). Uranium also has been
recovered from both fresh and sea water by Streptomyces vindochromogenes and
Chlorella regularis trapped in polyacrylamide (5).
Another alternative IS to use slurries of nonviable, killed biomass preparations entrapped in column reactors. Such preparations have previously been
shown to be effective metal accumulators (67). A further practical possibility
may involve the use of crossflow (tangential flow) membrane filtration, which
IS widely used m cell separation and processmg. Crossflow microfiltration
(CFMF) has particular advantages m that rt operates at low pressures and with
hrgh flux rates. Crossflow systemshave been demonstrated to be useful m harvesting and maintaining S. cerevisiae cells and cultures (8,9).
The procedures described m this chapter outlme the rmmobihzatron of yeast
and algal cells m polyacrylamide crosslinked gels, the entrappment of nonFrom

Methods

tn Biotechnology,

Ed/ted by D Sheehan

Vol

Humana

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2 B/oremed/atron

Press Inc , Totowa,

Protocols

NJ

Duncan, Brady, and Wilhelmr

viable yeast cells in column reactors, and the use of cross-flow microftltration
to
rmmobrlrze and retam yeast and algal biomass. All these procedures can be successfully employed for the broaccumulation
of a wade varrety of heavy metals
from a number of wastewaters (see Chapter 15).

2. Materials
2.7. General
1. To limit metal contammatton, all aqueous solutions are prepared with ultra-pure
water (Mrlhpore Mtlh-Q purtfrcabon system) All glassware used 1s boroslhcate
glass, whtch has relatively low metal-cation-bmdmg
properties Glassware IS prepared for use by washing with detergent, rrnsrng, and then heatmg m a 1 1 solutton of 55% mtnc acid* water solution (80C 12 h), washing with ultrapure
water, and heat drying. Metal analyses are carned out by flame atomic absorption
spectroscopy
2. The wet-pressed Saccharomyces cerevlstae biomass used was supplied by Anchor
Yeast Ltd (South Africa) (production stram, approx 90% cell vrablhty) and
washed wrth 4 vol of detomzed water and centrifuged (IOOOg, 10 mm) before use
Starting cultures of Scenedesmus, Selenastrum, and Chlorella were obtained from
departmental cultures
3. PIPES (PIPES, Srgma, St Lams, MO) or HEPES (Hrghveld Biologtcal, South
Africa) buffers are generally used throughout because of their negligible metal
chelating properties.

2.2. Gel Immobilization

1 Acrylamtde, N,N-methylene-blsacrylamtde,
N,N,N,N-tetramethylethylenedlamme (TEMED), and ammonium persulfate are available from Sigma. Analar
grade metal chlorides (Merck, South Afrrca) were used in broaccumulation studies
Effluents were obtamed from mdustnal sites

2.3. Entrappment

of Nonviable

Biomass

1 Analar grade NaOH and metal chlorides are available from Merck and Whatman
No 1 filter paper 1s used m all ftltratron steps.

2.4. Crossflow

Microfiltration

1 Polypropylene hollow fiber mlcrofrltratton membrane cartndges (membrane area

2 1 x IO- m2, pore size 0 1 PM) are available from the Polymer Research
Institute, Stellenbosch Umversity, South Africa Tns-HCl buffer, pH 7 2,ts used
to suspend cells

Bioremediation

of Heavy Metals

93

3. Methods
3.1. General
3.1.1. Yeast Cell Biomass Preparation
1. S. cerevzszae cells are washed twice with ultrapure water after centrifugatton at
IOOOg for 10 mm and resuspended in 5 mmol/L PIPES buffer which adjusted to pH
6 5 with tetramethylammonium hydroxide (TMAH, Sigma), or in Tris-HCl buffer,
pH 7.2

3.1.2. Algal Culture Maintenance

1 Isolates of the three spectes are obtamed by the spray plate method m whtch filtered
air is forced mto a vessel contammg an algal culture, whtch consequently propelled
the medmm out of a fme outlet (in this case a Pasteur pipet). The resultant aerosol
IS directed onto BG 1I-agar plates The plates are incubated at 22C on a light table
of 165.4 pE.rn2s light intensity. Workmg cultures of each species are grown from
a smgle colony.
2 All cultures are grown m BG 11 medmm pH 7 4, prepared according to Allen (10)
with ultrapure water (Mini-Q). The medium has low turbidity, allowing for penetratton of light reqmred by the photosynthetic algae The medium is sterthzed by
autoclavmg at 12 1C for 15 mm. The medium 1s stored at 4C until inoculated The
three species (one from each of the genus of Scenedesmus, Selenastrum, and
Chlorella) are harvested m the log phase.
3 The cultures are scaled up by adding 200 mL of the moculum to 2 5 L of BG IL
medmm. Samples are taken every 48 h using sterile glass ptpets, which are used for
cell counts and contammation checks. When the cultures reach the stationary phase
they are harvested and used for metal ion bioaccumulation experiments Duplicate 5
mL samples are drawn from the stationary phase cultures and filtered with Whatman
glass fiber (GF/A) filter disks through a Venturi pump. The filters are dried at 30C
overnight and weighed to determme the cell mass per volume of culture

3.2. Gel Immobilization

The method of immobilization used IS as follows: 10 g wet mass of S. cerevisiue is suspended in 20 mL physiologrcal saline (0.15M N&l) at 8C: acrylamlde monomer (7.5 g) plus NJ-methylene-bisacrylamtde (0.4 g) dissolved in
24 mL deionized water and cooled to 8C: The monomer solution and cell suspension are thoroughly mixed together. Added to this mixture IS 1 n-k of 2.5%
N,N,iV,iV-tetramethylethylenediamine (TEMED) and 5 mL of 1% ammomum
persulfate. The temperature of the solutionkuspenslon is maintained below 50C
during the exothermic polymerization process so as to not damage the biomass.
The immoblhzed biomass IS passed through a 30-mesh (500 pm) sieve. This
yields thin threads of yeast-cell-containing polyacrylamrde gel, 5 g wet mass of
which is subsequently placed m deionized water and then poured as a slurry mto

94

Duncan, Brady, and Wilhelm1

a chromatography column The flow rate is 1 mL/mm, the fraction volume 10 mL,
column height 10 cm, column volume, 20 mL, and temperature 20C f 2C.
Stock solutions of metal chlorides (200 ymol/L) or wastewater are passed
through the columns until the breakthrough point is reached (200-400 mL
depending on the metal, with 95-100% accumulation up to this point)
Desorption

of all metals except Co+ is achieved by passing lo-20

mL 0 1M

HCl, collected in 5 mL fractions, through the columns (90-100% desorption was

achieved). Columns

can subsequently

be reconditioned

by elutmg with 20 mL

0.0544 NaOH and 20 mL water, after which metal solutions can be reapplied.
3.3. Enfrappment

of Nonviable

Biomass

Wet yeast biomass (10 g) is mixed with 100 mL 2M NaOH and the solution

heated to 70-90C for 15 mm. The product is filtered through Whatman No 1

filter paper, washed with deionized water, and filtered again. The product is dried
on grease-proof paper at 70C for 12 h, milled to a gritty conststency,and passed
through a mesh (500 mrcrons, 30 mesh) to yield granular biosorbent.
Granular blosorbent (5 g) is loaded into 30 mL plastic columns and metal
solutions at 100 mg/L are pumped upward through the columns using a pert-

staltic pump at a flow rate of 25 mL/h (contact time was approx 60 mm) and the
temperature

is maintained

3.4. Crossflow

at 20C.

Microfiltration

Microfiltration membrane cartridges are made hydrophilic by the addition of

a small volume of surfactant (Teepol). Yeast and algal cells are immobilized on
the microfiltration cartridges m one of two ways:
1. Cells (14 g wet mass) are suspended for 10 mm at 25C m a reservoir containmg
200 pmol of the metal to be accumulated m 1 L of Tris-HCl buffer, pH 7.2 The
cells are harvested onto the membranes by pumping the cell suspension (by a pertstaltrc pump) from the reservoir around the exterior of the hollow fibers with the
permeate passing mto the lumen. The mfluent pressure 1smamtamed at 20 + 5 kPa.
Further addition of 200 pA4 metal solution can be maintained until breakthrough
(metal saturation of the biomass) 1s reached (generally 3 5-5 L). Fracttons (100
mL) of the permeate are collected for metal analysts
2. Cells (14 g wet mass) are suspended m Tris-HCl buffer (1 L) and pumped into the
filter cartridge as described above Immediately after this Trts-HCl, pH 7 2,
buffered metal solutron (200 PM) or wastewater is pumped through the
biomass-laden membranes until breakthrough was reached. Conditions are identical
to those described above.

In both casesdescribed above, after biomass saturation with metal, the metal
can be desorbed and recovered by pumping 50 mL of O.lM HCl through the

Bioremed~at~on of Heavy Metals

95

membranes, or the cells can be backwashed out of the membrane by reversmg

the flow and pumping water or buffer through the membranes from the lumen
into the exterior of the hollow fibers. In the former example, biomass can be
regenerated as described in Section 3.2. and in the latter case, new cells can be
loaded onto the membranes by either of the above methods. Serial microfiltration, using two or more hollow fiber membrane cartridges, is also possible using
the configuration depicted m Fig. 1.
Cells (14 g wet mass) are suspended in a 2 L solution of 200 FM metal
buffered with 5 mM Tris-HCl buffer, pH 7.2, or 2 L of wastewater. After 15 min
reaction time, the cells are harvested onto a hollow fiber membrane cartridge
(system I) restricting the reject flow. Constant additton of further metal solutron
(200 r-LM)maintains the mfluent reservoir at its initial volume (2 L), whereas the
permeate (100 mL/mm) is passed into a second reservoir (0.5 L), to which 14 g
wet mass of cells is added. The second cell suspension and subsequent permeate from system I is pumped onto a second membrane cartridge (system II),
which is similar to the first. Therefore, the effluent from the first accumulation
system 1sused as the influent for the second system and the permeate rates of
the two systemsare constantly matched. During filtration the cells pack onto the
membranes.
Volumes of 20-30 L of metal-containing solution can be processed m this
manner. Metal-saturated biomass is removed sequentially from each cartridge as
previously described and is desorbed of metal as described in Section 3.2. This
system allows for processing of large volumes of effluent and also facilitates the
selective accumulation of different metals in a mixed metal solution since there
is a preferential or partially preferential binding of specific metals m each cartridge resulting from differences in affinity of the biomass for different metals. It
is also possible to load different forms of biomass in each cartridge that may further facilitate preferential metal accumulation in the individual cartridges.
4. Notes
I. PIPES and HEPES buffers are expensive for large scale use. Cheaper buffers (such
as phosphate buffers) can be employed where a small amount of metal chelation by
the buffers is not cructal. In wastewater treatment buffers are not employed
2. The processes descrtbed m this chapter can be used with any form of biomass.
3. Some loss of biomass does occur durmg the processmg of heavy metal contammg
water, but this is negligible Loss of biomass can be followed by measurmg the
increase m absorbance of the effluent at 540 nm.
4. pH affects the rate and extent of bioaccumulatton. For most metals bioaccumulation generally decreases at reduced pH.
5 Immobihzation of biomass considerably improves the level of btoaccumulation
when compared to batch reactors.

96

Duncan, Brady, and Wilhelmi

Permeate
Reject
rlII

Fig. 1. Schematic representation of the serial mlcroflltration

tion. I and II represent the membrane cartndges.

equipment configura-

Reuse of biomass after desorptlon and recondltlonmg is possible for 5-10 times
before efficiency 1s affected.
Polyacrylamlde lmmoblllzatlon 1s found to be superior to calcium algmate, glutaraldehyde, agar or cellulose-acetate lmmobihsation Polyacrylamlde lmmoblhsatlon has the advantage that its not prone to damage by cation replacement or
chelation as calcmm-ahgnate systems are-an important attribute when accumulatmg metal catlons Moreover, algmate systems are unstable at high pH The hmltatlon of the gel lmmoblhzatlon IS the cost of the gel if used on a large scale and the
engineering of suitable columns to contam the gel Rates of diffusion through the
gel may also become hmltmg
The advantages of nonviable cells are numerous Killed cells may be stored or
used for extended periods at room temperature without putreficatlon occurring
Moreover hvmg cells are prone to the toxic effects of effluents, which may
result m cell death, thereby negating any of the advantages of utlllzmg live cells
Some methods of klllmg cells may actually improve biosorption properties of the
biomass
Hollow-fiber
crossflow mlcroflltratlon based bloaccumulatlon systems are potentially less expensive than gel lmmoblhzed biomass and allow for more rapid
adsorption and desorptlon of metals. They are also systems that can feasibly be
engineered on a large scale

Bioremedia tion of Heavy Metals

97

10 The crossflow mtcroftltratron processes may also employ membranes m the form
of tubular or rolled flat sheet systems. They may be reverse osmoses or ultraftltranon membranes and may also be manufactured of polysulfone, polycarbonates, or
polyvmyl chloride.
11 Contmual reuse of biomass zn situ on the membranes may result in the formation of
broftlms. This may alter the broaccumulation process but is unlikely to affect the
overall efficiency of the system.

References
1. Brady, D and Duncan, J. R. (1994) Btoaccumulatton
of metal cations by
Saccharomyces cerevulae. Appl. Mzcroblol Bzotechnol 41, 149-154
2 Lewis, D. and Krff, R J. (1988) The removal of heavy metals from aqueous effluents by tmmobrhsed fungal biomass Environ. Technol. Lett 9,991-998
3 Tzezos, M., McCready, R G L., and Bell, J. P (1989) The contmuous recovery of
uranium from btologtcally
leached soluttons using tmmobthsed
biomass
Bzotechnol Bioeng 34, IO-17
4 Tsezos, M. (1984) Recovery of uranium from btological adsorbents-desorptton
equlltbrmm Blotechnol Bcoeng 26,973-98 1
5. Nakajima, A , Honkosht, T , and Sakagucht, T. (1982) Recovery of uramum by
rmmobrlised mrcroorgamsms
Eur J Appl Microbial Blotechnol. 16,88-91
6. Brterley, J A., Brterley, C. L , and Goyak G. M. (1986) AMT-BIOCLAIM.
a new
wastewater treatment and metal recovery technology In. Proc. of the 6th Zntl
Symp. Blohydrometallurgy, Lawrence R W. et al. (eds ), 29 L-304
7. Fourest, E. and Roux, J -C (1992) Heavy metal btosorptton by fungal mycehal byproducts mechanism and influence of pH. Appl. Mlcroblol
Blotechnol , 37,
399-403
8. Urrbelarrea, J -L , Winter, J , Goma, G , and Parerlleux, A (1990) Determmatron of
maintenance coefftctents of Saccharomyces cereviszae cultures with cell recycle by
cross-flow membrane filtration Bzotechnol Bloeng. 35,201-206
9. Warren, R. K , MacDonald, D. G., and Hill, G. A. (1991) Cross-flow mtcroftltratton of Saccharomyces cerevislae. Proc Blochem 26,331-342
10 Allen, M. M (1968) Simple condtttons for growth of unicellular blue-green algae
on plates. J Phycol 4, l-4