725

Entrapment of particles from suspensions using Aspergillus species

K. M. Paknikar*, J. M. Rajwade and P. R. Puranik

Division of Microbial Sciences, Agharkar Research Institute, G.G. Agarkar Road, Pune
411 004, India

A fungal isolate belonging to Aspergillus sp., could entrap different particulates such as
ferric oxide, granulated carbon in pellets during growth. The entrapment of suspended
particulates was dependent on two factors, viz. ability of the particulates to remain in
suspension and the growth morphology of the fungus. Growth morphology of the fungus was
found to be governed by composition of medium, inoculum size, incubation conditions etc. It
was possible to suitably modify these factors to achieve increased entrapment of the particles.
In experiments with iron ore, it was found that although the weight of ore entrapped in the
pellets increased from 590 mg to 640 mg with the increase in inoculum size from 1 ml to 5 ml,
the iron content of entrapped ore decreased from 56.5% to 43.0%. This indicated that with
increase in inoculum size and consequent decrease in the pellet size, less number of iron
particles were entrapped by the fungus although overall particulate entrapment increased.
These results indicated that this phenomenon could possibly be used in practice for (a)
removal of suspended particulate matter from waste waters and (b) beneficiation/upgradation
of ores by removing the impurities.

1. INTRODUCTION

Fungi are ubiquitous and may be even dominant in adverse environments such as metal-
polluted sites. They exhibit a variety of responses towards heavy metals (1) which, in principle,
could be exploited in developing biotechnological methods for the abatement of pollution
caused by metals. For example, biosorption of metal ions by fungal biomass has been studied
extensively (2). Fungi are known to bring about various kinds of metal transformations, viz.
methylation of selenite (3), reduction of chromium (4), oxidation of manganese, solubilization
of iron from iron oxide (5), etc. Fungi are well suited for use in transformation of metals as
they have an intrinsic ability to tolerate high concentrations of metals and extreme pH
conditions (6). Many filamentous fungi can adsorb particulates such as elemental sulfur,
insoluble sulfides, charcoal, clays and magnetites (7,8). As early as 1918, Williams (9) showed
that fungi could adsorb gold from colloidal solution, although this ability seems not to have
been exploited industrially. During our recent studies, an Aspergillus sp. was found to possess
an extraordinary ability of entrapping particulates from solutions inside the pellets

"Corresponding author, Fax: +91-20-351542, E-mail: paknikar@vsnl.com

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during growth. When the fungus was grown in a ferric oxide containing medium, particles in
suspension were entrapped in fungal pellets thereby clarifying the medium. It is well known
that certain physiological and physico-chemical factors influence pellet morphology of fungi
(10). Since such an entrapment property could be potentially used in clarifying effluents by
removing insoluble materials, an attempt was made to investigate the phenomenon further and
the results obtained are presented in this paper.

2. MATERIALS AND METHODS

2.1. Microorganisms and growth conditions

In our earlier studies on the heterotrophic leaching of iron a fungal culture was isolated from
municipal sewage sample. The strain was identified as Aspergillus sp. and was deposited in
MACS Collection of Microorganisms (World Data Center Code No. 561, Accession No.
MCM F-2). The strain was maintained on Sabouraud's agar slants and stored at 5~

2.2. Effect of medium composition and inoculum size

During the experiments the fungus was grown on Sabouraud's agar at 30~ and the spores
were harvested from 5 days old slants. They were suspended in sterile 1:1000 triton-X 100
solution. Density of the suspension was adjusted to 1 x 108 spores/ml determined by a
microscopic count.
In order to study the effect of growth media components on pellet morphology, the spores
were inoculated in a series of 250 ml Erlenmeyer flasks containing various combinations of
glucose (1-25 g/l) and peptone (5-20 g/l). pH of the medium was adjusted to 4.0 and medium
to flask volume ratio was maintained at 1:5. Flasks were incubated at 30~ on a rotary shaker
(Gallenkamp, UK) at 120 rpm for 72 h. After growth, pellets were observed microscopically
and their sizes were determined using a stage slide micrometer. The utilization of glucose
during growth was assessed by dinitrosalicylic acid (DNSA) method. Dry weight of the fungus
in the experimental set was also determined. For determination of dry weight, fungal mycelia
were harvested by filtering through Whatman No. 1 filter paper, dried in an oven at 60~ and
dry weights were recorded.
Effect of inoculum size on the pellet morphology was checked by inoculating a series of
flasks containing 50 ml Sabouraud's broth with different volumes of spore suspension (1-5
ml).

2.3. Entrapment of particulates by Aspergillus sp.

The ability of the Aspergillus sp. MCM F-2 to entrap particulate matter was checked in
Sabouraud's medium supplemented with sterilized particulates (viz. granulated charcoal, sand,
ferric oxide, manganese dioxide, a combination of ferric oxide-manganese dioxide, iron ore) at
1% w/v level. All the samples had a particle size distribution in the range of 100-150 mesh
(0.149-0.105 mm) according to Tyler standard screens. Flasks were inoculated with 1 ml
spore suspension and incubated at 30~ for 72 h. After incubation, usual growth parameters
were measured. Fungal pellets containing the entrapped particles were separated by sieving
and their dry weights were recorded. In case of experiments involving iron ore, samples of the
pellet-entrapped ore and the residual ore in flasks were digested in minimal amounts of
concentrated HC1. These samples along with the spent media were analyzed for total iron
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content by using an atomic absorption spectrophotometer (Perkin Elmer, Model 2380, USA).
The changes in pellet structure during growth in presence of ferric oxide was checked using an
electron microscope (Stereoscan 200, Cambridge Instruments, UK). For this purpose, pellets
grown in presence/absence of ferric oxide were fixed in 2% glutaraldehyde for 18 h and
fixation was followed by dehydration using 5-100% ethanol. All procedures were carried out
at 4~ The samples were coated with platinum (Biorad, UK). In case of flasks containing
ferric oxide scanning electron microscopic observations were made.

3. RESULTS

It was observed that by varying glucose and peptone concentrations in the media, growth
morphology of the Aspergillus sp. MCM F-2 changed considerably (Table 1). At glucose
concentration of 10 g/l, dry weight of the fungus increased in response to increasing
concentrations of peptone (5-20 g/l). A similar observation was made with fixed peptone
concentration (20 g/l) and varying glucose concentrations (1-10 g/l) in the media. Data in
Table 1 further show that cultures containing 10 g/1 glucose, and peptone concentrations in the
range of 5-15 g/1 had clumped, coalesced growth. With peptone concentration of 20 g/l,
however, a pelleted growth was obtained. Residual sugar data demonstrated that very little
glucose was utilized in cultures containing lower peptone concentrations, which indicated that
growth in these cultures was limited by nitrogen levels. It was further observed that inoculum
size had a marked effect on the average pellet size when pelleted growth was obtained. It

Table 1
Effect of peptone and glucose concentration on growth morphology ofAspergillus sp.

Medium
9composition Growth characteristics of Aspergillus sp.

Peptone Glucose Glucose Dry weight Growth Average pellet

(g/l) (g/l) (residual) of fungus morphology size
(g/l) (mg/100 ml)
Inoculum

1 ml 5 ml

10 20 17.05 621 4 Pelleted 2.0 0.5

20 10 2.76 1021 6 Sporulated pelleted 2.5 0.5
20 5 0.32 840 8 Pelleted 2.0 0.5
20 1 0.33 703 4 Pelleted 1.0 0.5
15 10 7.12 507 6 Coalesced growth -

10 10 5.26 449 0 Coalesced growth

5 10 9.66 128.0 Coalesced growth
5 25 22.37 315.0 Coalesced growth
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Table 2
Growth of Aspergillus sp. in the presence of particulate matter

Particulates used Growth characteristics ofAspergillus sp.

Activated charcoal Uptake of activated charcoal in pellets

Sand No uptake of sand, pelleted growth
Ferric oxide Uptake of ferric oxide pelleted growth
Manganese dioxide No uptake, pelleted growth
Ferric oxide + Manganese dioxide Uptake of only ferric oxide, pelleted growth
Iron ore Uptake of iron ore in pellets

could be seen that pellet size decreased proportionately with increase in the level of inocula.
Experiment carded out to check the ability of Aspergillus sp. MCM F-2 to entrap various
particulates (Table 2) showed that although pellet formation was observed in all the flasks,
there was no entrapment of sand and manganese dioxide. In case of manganese dioxide-ferric
oxide mixture, selective entrapment of ferric oxide was observed. When an iron ore was used,
uptake of the ore particles was observed.
The pellets from flasks supplemented with ferric oxide were red in color. Light microscopic
observations revealed presence of amorphous ferric oxide particles entangled in the fungal
filamentous structure. In order to assess whether the entrapped particulates had any influence
on surface topography, pellets were observed using a scanning electron microscope.
Observations revealed that diameter of mycelia decreased by almost 50% in the presence of
iron oxide (Figure 1). Further, the filaments in this case were tightly arranged as compared to
flasks without iron oxide.
When particulate entrapment experiment was carried out on a quantitative basis with an iron
ore by varying the inoculum size, interesting observations could be made (Figure 2). It was
~'ound that although weight of ore entrapped in the pellets increased from 590 mg to 640 mg

• ~0~%i~!i!iiiiii

Figure 1. Scanning Electron Micrographs of Aspergillus pellets before (le~) and after (fight)
entrapment of ferric oxide. Note reduction in mycelial diameter and increased compaction in
ferric oxide entrapped pellets (right) (Magnification 1000X).
 729

58 700
54 660

620 i
a~ 46 580

~ 42 540
38 500
1 2 3 4 5
SPORE SUSPENSION, ml
IO % Fe in entrapped ore 9 wt. of entrapped ore I

Figure 2. Effect of inoculum size on entrapment of iron by

Aspergillus sp. from iron ore.

with increase in the inoculum size from 1 ml to 5 ml, the iron content of entrapped ore
decreased from 56.5% to 43.0%. This indicated that with increase in inoculum size and
consequent decrease in the pellet size, less number of iron particles were entrapped by the
fungus although overall particulate entrapment increased.

4. D I S C U S S I O N

Results obtained with Aspergillus sp. MCM F-2 showed that composition of medium
significantly affected growth morphology of the fungus. This result suggested that
pellet/filament form could be obtained by changing proportion of the media constituents.
These results were in agreement with those observed for Rhizopus arrhizus by Byrne and
Ward (11). The pelleted morphology is desirable in certain industrial applications such as
antibiotic fermentation, vitamin production, etc. However, filamentous morphology is
preferred in industrial production of organic acids.
Wainwright et al. (7,8) reported that mycelium of Mucor flavus adsorbed calcium silicate,
elemental sulfur, lead sulfide and zinc dust from suspension. It also adsorbed ferric hydroxide
from acid mine drainage. The adsorption increased with increase in proportion of mycelium
and addition of a carbon substrate proved inhibitory to the process. This phenomenon can be
described as "bioadsorption". However, entrapment of particulates described in this paper is
different from the above observation because particulates were not adsorbed sensu strictu but
were probably enmeshed in the tight mycelial structure of the pellet. Since pellet formation
was influenced by medium composition, the entrapment was also affected by these factors.
Entrapment of suspended particulates is a phenomenon dependent on two factors, viz. the
ability of the particulates to remain in suspension and growth morphology of the fungus. These
factors, in turn, are govemed by various physical and physiological properties like specific
gravity of the particulates, composition of growth medium and growth conditions. Since
medium composition and the growth conditions for particulate entrapment experiments were
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identical, it could be inferred that ability of the particulates to remain in suspension was a
significant factor affecting entrapment. Our studies do not answer the mechanism of
entrapment fully at this stage. However, they point out a new area in mineral biotechnology
which could possibly be exploited for (a) separation of suspended particulate matter from
waste waters and (b) beneficiation/upgradation of ores by selectively removing the impurities,
when conventional methods are not feasible.

REFERENCES

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Academic Press, London, 1987.
2. A. Kapoor and T. Viraraghavan, Biores. Technol., 53 (1995) 195.
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Biohydrometallurgical Technologies Vol. II, The Minerals, Metals & Materials Society,
Warrendale, PA, 1993.
5. J.C.G. Ottow and A. Von Klopotek, Appl. Microbiol., 18 (1969) 41.
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