779

Transformation o f arsenic and tellurium in solution by fungi

P.M .Solozhenkin~, V. P. Nebera b and N. N. Medvedeva-Lyalikova~

aInstitute of the Problems of Complex Utilization of Mineral Resources RAS,

Kryukovsky tupik, 4, Moscow, E-20, Russia, 111020

bMoscow State Geological Prospecting Academy,

Miclucho-Maklaya, 23, Moscow, Russia, 117873

r of Microbiology RAS,
Prospect 60-1etiya Oktyabrya, 7, korp. 2, Moscow, Russia, 117811

The fungus Scopulariopsis brevicaulis was studied for treatment of arsenic-bearing

solutions. The fungus changed dissolved arsenic AsO43 into the gases, arsine and
trimethylarsine. From 93 to 99% of the arsenic were recovered under optimal conditions. The
gases were transformed into high quality arsenic by thermal treatment. The fungus Penicillium
chrysogenum was studied for reduction of tellurium to elemental tellurium and the gas
dimethyltelluride. The fungus was used to extract tellurium from electrolytic slimes of
complex composition in copper refining. From 89 to 98% of tellurium were extracted.

1. INTRODUCTION
Bacterial leaching is adventageous in the xtraction of arsenic from different materials.
Optimal conditions for dissolution of arsenic have been determined [1-4]. The recovery of
arsenic from solution remains a difficult problem. Existing recovery methods, which envolve
arsenic immobilization, can be divided into three groups [5]: 1) sorption using activated coals,
aluminates, clays, titanium-based materials, or metallic sorbents (Fe, Zn), 2) precipitation
reactions, including thermal precipitation of As, coprecipitation, precipitate flotation, sorptive
colloidal flotation, sulfide precipitation of arsenic, or formation of calcium magnesium
arsenate Fe(III) and AI(III), 3) other technologies, including electrolysis, solvent extraction,
diffusion membrane screaning, ion flotation, chemical redox processes, and biological
processes.
Regardless of which of the above treatment are used for recovering arsenic, certain
environmental conditions may cause some re-solution of immobilized arsenic. In none of the
technologies cited above is the immobilization of arsenic absolutely irreversible. New
processes need to be developed for arsenic extraction so that immobilized aresenic in the
elemental state or as arsenic(V) sulfide, can not subsequently polute the environment. We
consider less common microbiological methods of arsenic immobilization preliminary.
The fungus ScoFulariopsis brevicaulis id known to possess an ability to reduce arsenic and
transform it into trimethylarsine, a gas [6,7]. This characteristic of fungus is exploited for
removing arsenic from liquids [8,9].
Choice of ways for cleaning of industrial sewers and sewages from harmful admixtures of
tellurium depends on their qualitative and quantitative compositions, as well as from required
780

degrees of cleaning and possibility of using an extracted metal. Full recovering of tellurium
from solutions may be achieved by coprecipitation with aluminium hydroxide. When tellurium
is cemented, and then reduced, 93% of it can be recovered [ 10]. For purification of tellurium a
number of researchers favor thermochemical process (destillation) because less material is
needed, labour costs are lower, and recovery is faster [11 ].
A process for removal of tellurium from industrial wastes exists and is needed. However,
there is a problem in removing tellurium from sewers microbiologically, which resides in
initial atage of the process. Insufficient attention ahs been paid to micelial fungi, though they
are known to grow rapidly and have a great potential for forming different useful metabolic
products, and their biomass has great sorption capacity [ 12,13]. Lower fungi are able to reduce
tellurium and selenium to their elemental state [14]. This fungal traits have been used by the
authors in the extraction of tellurium from solutions and from various ore treatment wastes. Of
all fungi tested, the most tellurium-tolerant was a culture of the fungus P. Chrysogenum. Its
most useful traits were its ability to reduce oxidized tellurium compounds and to methylate
tellurium [ 14,15].

2. MATERIALS AND METHODS

2.1. Organisms and enriched cultures

The fungi, Scopulariopsis brevicaulis and Pennicillium chrysogenum, used in this study,
were obtained from the National culture collection in Moscow. They were routinely grown in
slightly modified liquid Chapek's medium. To make cultivation inexpensive, complex media
with all the nutrients required by the fungi were used [16]. To stimulate growth, microbial
phospholipides (FMF), cotton seeds extract (ECS), an aqueous extract of weathered brown
coal (CE) were added to Chapek's medium. These suplements decreased tyhe consumption of
sucrose (50%) in the medium. CE substitudes all mineral salts. CE contained 50-70% organic
materials and 30-50% mineral salts. The organic component of CE contained (in %): C 24-29,
H 4.0-4.5, and (N+0) 66-72. The chief mineral components of CE were Si, AI, Mg, and Mn.
These medium modification enhanced biomass accumulation and speeded up arsenic
transformation. The stimulatory effect of CE is attributed to the presence of physiologically
active compounds in it, such as ferric-, magnesium-, calcium- as well as silicon-organic
compounds [13]. They stimulated the early developmental stages of the fungi by halving the
length of the lag phase. The additives accelerate the remediation of arsenic-containing
solutions and thereby reduce the primary treatment cost.

2.2. As and Te samples

Test were performed with various solutions with different concentrations of arsenate from
thiobacillus bacterial leaching, production effluents from Alawerdsky mining-metallurgical
complex, containing arsenic with different degree of oxidation. Were also used electrolytic
slimes from copper refining, containing, %: Te 0.6-1.0; Sb 1-3; Pb 0.5-1; Fe 0.5-1; Cu 2-7;
CaO 0.7; MgO 0.3-1.
 781

3. RESULTS AND DISCUSSION

3.1. Inoculum preparation, incubation, metal ions tolerance of the fungi

3.1.1. Scopulariopsis brevicaulis

Non-adapted microorganisms, in particular when working with the ore raw material, are
influenced upon their vital activity by different cations and anions [ 10]. We therefore studied
adaptation of Sc. hr. to cations and anions in various arsenic-containing sewage samples.
Adaptation of the fungus Sc. hr. to mercury, stibium, copper, lead, cadmium, and cobalt was
realized by the resowing on Chapek's agarized medium with gradual increasing the
concentrations of cations. The accumulation of biomass and radial growth rate of fungi was
followed at the different ion concentration. Adaptation of the fungus Sc. br to cations
extracted by the water from the arsenic containing ore, and after leaching arsenic by thio
bacteria from the gold- stibium ore were also studied. The ore was wet-grinded to 80 % of
class - 74 lxm and filterred. The filtrate was sterilized before addition to the growth medium,
with following addition of the cations (Table. 1).
The sodium arsenate solutions with various concentrations As or sewers of Alawerdsky
mining metallurgical combine, containing arsenic with different degree of oxidation, were
used.
From data of Table 1 follows, that presence of heavy metals cations oppressed fungi
growth. Ore extract improved vital activity of fungi. This difference may have to do with the,
presence of biologically active materials in the extract.

Table 1
Influence of metal cation concentrations on radial growth of fungi (Arsenic contents - 1.5 g/l,
duration of ~ o w t h - 5 days)
Cations Average diameter of fungal colony, mm
concentration, Hg Pb Cu Sb Co Cd
mg/1
0 54.0 54.0 54.0 54.0 55.0 54.0
25 16.5 18.0 14.5 16.0 17.0 10.5
50 12.7 12.4 7.6 10.4 10.0 6.0
100 4.0 8.5 4.5 0 0 0
200 0 3.0 4.2 0 0 0
600 0 0 0 0 0 0
Extract from arsenic-containing ore
0 55.0 54.0 54.0 54.0 56.0 57.0
12 30.4 42.4 40.5 34.5 28.5 24.0
25 25.0 36.0 24.0 26.0 21.5 20.0
50 12.5 16.5 19.5 21.0 10.4 0
100 5.0 11.0 14.0 16.0 0 0

Table 1 shows that as the fungal mass increases, arsine + trimethylarsine (gas) production
increases. When the initial arsenic concentration is too high, gas production by Sc. Br. is
depressed. It was possible to increase the arsenic tolerance of the fungus to increased
782

concentrations of arsenic to 10 g/l. The adapted to high concentrations of arsenic fungus Sc.
Br. produced significant quantity of biomass, however gas production was three to four times
slower that at low As concentration.

3.1.2. Penicillium chrysogenum

To make the reduction of dissolved tellurium by P.chrysogenum inexpensive, 2-6 % of
waste-liquor from acetyl-butyl fermentations (ABF) from Grozny were added to Chapek's
medium. Organic compounds in the ABF replaced expensive sucrose in the medium. Table 2
shows how biomass yield depends on concentration of various medium supplements in arsenic
removal.

Table 2
The role of medium supplements in biomass yield and arsenic removal (initial concentration
of arsenate - 5 g/l)
Length of fungal Yield of fungal Recovery As in gas, Comments
growth, days biomass: dry mass, % %
to control
Control optimal conditions Lower fungus was
3 100 99.5 grown on Chapek's
medium
1 60.4 84.4 For inactivation of
3 81.1 80.9 fungal growth
6 92.2 87.7 corrosive sublimate
9 98.8 96.8 was used (0.2 mg/l)
1 140.0 98.6 For activation of
3 186.4 99.4 fungus growth extract
6 121.4 98.1 of brown coal was
9 r
111.4 96.5 added (0.2 mg/l)

The fungus was adapted to increasing concentrations of tellurium by consequent resowing

on agarized Chapek's medium with gradual increasing concentrations of tellurium over a
range of 0.5-7.5 g/dm 3. The fungus was also adapted to arsenic-containing ore by cultivation
in ore extract produced by soaking 1 part of ore, ground to a particle size 90% -0.074 mm in 3
parts of water during 24 hours, with following filtrations. In addition, the fungus was adapted
to increased contents of rig, Sb, Pb, Cu, Co, Cd by consequent resewing on agarized Chapek's
medium, prepared on the ore extract with gradual increasing of respective cation
concentrations (Table. 1). Table 3 shows, that presence of heavy metals cations inhibits
development of fungus, however, in the ore extract, growth of fungus was intensified, that
bound, much more likely, with presence of biologically active materials required for fungal
growth.
 783

3.2 Production of fungi mutants

3.2.1. Ultra-violet irradiation o f Scopulariopsis brevicaulis

Fungus cultures were irradiated with bactericidal lamp (DB-30).
Mutants of Sc. Br. With enhanced arsine + trimetilarsine (gas) producing capacity were
induced by ultra-violet irradiation [ 16]. Survival of irradiated fungus were defined by method
of macro colonies. Under repeated UV-irradiating of the fuingus colonies vere selected
mutants, capable to translate arsenic in gaseous form quicker than inicial species. As can be
seen from the figure 1, the fungus survived under significant length of irradiation.
Yield of fungi biomass growing on the medium, containing 40-50 % standard Chapek's
medium and 60-50% dissolve, containing 4% ABF, increased by 6-8 times in contrast with
control. Introduction to the media a departure of shuger production - molassa (3 % of sugar),
sodium nitrate (2 g/dm 3) and mono-substituted potassium phosphate (1 g/dm 3) have allowed to
enlarge yield ofbiomass more then 3 times.

ot~ 50
o E
E
0
2
~m

o o. 3

o 9~- 5
9 #
o

>
I

$ t0 15 g0 Z~-
3 30 a~ ~r, s 1", days

Fig 1. Survival S. brevicalis under different Fig.2. The radial growth of fungus on
duration of UV-irradiation: 1-Chapek's Chapek's medium with arsenic at different
medium; 2- Chapek's medium with duration of UV irradiation. Mutants:
arsenic-containing solution. l-N8; 2-N7; 3-N4; 5-N6; 6-N5.

Under short UV-irradiating (<30s) presence of the arsenic in the medium renders
stimulating action. This may have been due to production of secondary metabolites that help
to protect the fungus against arsenic toxicity. Asenic toxicity was assessed by measuring the
rate of radial growth. The growth rates and biomass production of different mutants differed
(Figure 2.).
784

sd0
80

lip
o
o ,O
rv,

IV I 1 I
0 2,,,q" 5,0 TI~, g .l'

Figure 3. Tellurium recovery from solutions depending of growth

method: 1 - inoculation; 2 - eddition of pregrown fungal biomass.

3.2.2. Ultra-violet irradiation of Penicillium chrysogenum

Fungus cultures were irradiated with bactericidal lamp (DB-30). The mutants selected after
irradiation produced twice as much biomass and exhibited a shorter lag phase, as compared to
initial culture

3.3. Arsenic removal by Scopulariopsis brevicaulis

Arsenic removal from waste waters and sediment from Alaverdsky plant, containing in
rag/l: 3.8 As; 710 Fe; 0.91 Mo; 1.18 Zn, 1.04 Pb; 341.67 C, and having pH 3.4 was tested. To
optimize fungal activity, the pH of waste water was brought to neutrality and the effluents
were then filtered. The filtrate and the sediment contributed a culture of fungus for the
following tests. The intensifying additives - phosphatides of microbial fat were added (0,2
rag/l). In 6 days, the fungus converted 99.5% of the dissolved arsenic and 97.7 % of arsenic
from the sediment to arsine + trimethylarsine.
The observed arsenic removal became the basis for making an installation [8,9].The
reactor was a round-bottomed glas flask, in a thermostatic water bath. The reactor was filled
with arsenic-containing solution and nutrient medium and inoculated with the fungus. The
system had hydrogen gas passed trough it. After 5 days of fungus growth the arsine +
trimethylarsine produced by the fungus was blown off and collected in a trap. Carrier gas was
than used to transfer the arsines to a furnace for combustion at 560~ On the way to furnace
gas was cleaned from the hydrogen sulfide with the lead acetate and dried by passing trough
calcium chloride. Thermal decomposition of the arsines led to formation of elemental arsenic
of high-quality with commercial value. The reaction in the furnace can be presented as
following:
2(CH3)3As = 2As + 6C + 9H2 (1)
The residual gas is passed into a receptacle with potassium permanganate for final
cleaning.

3.4. Purification of industrial effluents from arsenic using sulfate-reducing bacteria

(s~)
Many examples of removing heavy metal ions from industrial effluents with hydrogen
sulfide generating by SRB can be found in publications [ 18-20]. Thus, Cu, H~ Pb, Co, and Cd
 785

can be removed from waste water. In the case of As removal, As(Ill) is first oxidized to As(V)
being less toxic, than As(Ill). The As(V) is then precipitated as arsenic sulfide with hidrogen
sulfide generated by SRB.
From the dilute solutions, containing As less that l g/dm3, last can be removed by
biosorption using spent biomass from antibiotic production. Such bioremediation techniques
are useful ecologically by immobilizing toxic metals temporarily or permanently.

3.5. Recovery of Te with Penicillium.chrysogenum

The water extract of weathered brown coal (CE) was used as intensifying additive for
increasing the yield of fungal biomass. The growth of fungus P. chrys., adapted to 5 g/dm3 Te
was stimulated two-fold by the addition of CE. At a concentration of CE 0.1 g/dm3 the time
required for removal of tellurium from solution decreased from 16 to 9 days and tellurium
recovery increased from 89.8 to 98.5%.
Two methods of tellurium immobilization were studied: 1) direct dissolution with a spore
suspension of the fungus; 2) dissolution by pregrown biomass. The second method was more
efficient than the first. (Figure 3). The following factors may influence the results: lack of
grown inhibition of fungi activity by the presence of cation that increased a possibility to
sorptions of tellurium on the biomass of fungus and realization of fermentation reactions.
Reduction of tellurium requires living biomass. Telurium recovery from solution requires
its reduction to elemental Te and methylated tellurium, gaseous compound (15-20 %). The
methylated tellurium is, probably, dimethyltelluride, which can be sorbed on the natural
zeolite with particles size 0.5-0.1 mm (Dzegvi deposit).

Table 3
Reduction of dissolved Te by P. Chrysogenum (initial Te contents - 0.5g/l; duration of
experiment- 9days)
Contence of The amount of reduced Te Notes
biomass, ~1
Sum. gas metal
3.63 89.2 8.1 81.1 Pregrown
4.52 93.83 9.63 84.2 biomass added to
5.38 97.76 11.76 86.0 solution
6.23 98.8 15.7 83.1
7.60 98.4 22.4 77.0

3.71 68.80 6.4 62.4 Inoculation with

4.68 71.73 7.23 64.5 fungal spores
6.01 78.38 8.08 70.3
6.64 80.41 8.21 72.2
7.84 86.51 10.11 76.4

Received elemental tellurium was analyzed by physicochemical methods. Comparison of

characteristic peaks of X-ray spectra at 0,223, 0,234 and 0,323 nm for pure tellurium and
spectral characteristic of recovered product (admixtures AI, Ca, Mn, Fe, Sn in the amount 1-
10-5 % and admixtures Mg, Pb, Sb in the amount 1-10.3 %), showed that recovered tellurium
was technicaly clean product.
786

P. chr. only reduced tellurium from tellurium-containing materials, such as electrolytic

slime in particular. It did not act on other accompanying cations. Fungus strains that were
adapted to increased concentrations of some components of electrolytic slimes, containing, %:
Te 0.6-1.0; Sb 1-3; Pb 0,5-1; Fe 0.5 -1; C 2-7; CaO 0.7; MgO 0.3-1 exhibited enhanced activity
(Table 3).

4. CONCLUSION

It is posible to remove arsenic microbiologically from effluents of mining and

metallurgical complexes. Microbiological removal of tellurium from industrial waste streams
can substitute labor-intensive storage of electrolytic slimes produced in copper and lead-zinc
production, and result in the production of high-quality tellurium. Fungi can be successfully
used in mineral processing, hydrometaUurgy, and treatment of industrial waste streams. The
biohydrometallurgy of arsenic and tellurium and the characteristics of the microbiological
processes involved are important for assessing their techno-economic application in the
future. These recovery processes may lower the costs of collection, storage and recycling of
such materials and the treatment of vast quantities of industrial waste waters.

REFERENCES

1. G. I. Karavayko, S. I. Kuznetsov, A. I. Golomzik, The role of microorganisms in leaching

of metals. Moscow, Science, 1972.
2. S. I. Polkin, E. V. Adamov, V. V. Panin, The technology of bacterial leaching of non-
ferrous and rare metals. Moscow, Nedra, 1982.
3. G. G. Kulebakin, Bacterial leaching of sulfide minerals. Novosibirsk, Science, 1978.
4. A. N. Ilyaletdinov, Microbiological conversion of metals. Alma-Ata, Science, 1984.
5. V. Nenov, A. I .Zouboulis, N. Dimitrova, I. Dobresky, Environ. Pollut. 83 (1994).
6. Z. E. Becker, Physiology of fungi and their practical use. Moscow, MSU (1963).
7. D. Foster, Chemical activity of fungi. Moscow, Inostr. Literature, 1950.
8. P. M. Solozhenkin, L. L. Lyubavina, S. A Sherepova., N. N. Lyalikova, Recent Progress in
Biohydrometallurgy, G. Rossi & A. E. Torma (eds.) Associazione Mineraria Sarda, Iglesias,
Italy (1983).
9. P. vl. Solozhenkin, L. L. Lyubavina, N. N. Buyanova, Tsvetniye Metally, 6 (1987) 24.
10. A. A. Kudryavtsev, Chemistry and technology of selenium and tellurium. Moscow,
Metallurgy, 1968, 153.
11. L. A. Niselson, A. A. Titov, The reception and analysis of materials of high purity.
Moscow, Science, 1978, 98.
12. A. N. Ilyaletdinov, Trans. Internat. Seminar, Sofia (1982) 349.
13. W. J. Niekerson, W. A Taber, Y Falcone, Can. J. Microbiolog., 2 (1956) 575.
14. Y. Falcone and W. J. Nickerson, Enzymatic reduction of selenite. Bacteriol. Proc. (1960)
152.
15. P. M. Solozhenkin, L. L. Lyubavina, N. N. Buyanova and I. V. Kirshenina, Tsvetnie
Metally, 11 (1992) 71.
16. Yu. T. Lyakov, Science (1980) 45.
17. Z. A. Avakyan, Itogy Nauky i Techniky, Microbiology, 2 (1973) 96.
18. F. G. Vafina, Z. A. Rumyantseva, Z. I. Pevzner, Gumine fertilizers and their using, Theory
and practice, v. IV, Dnepropetrovsk, Agrycultural Inst., 1980, 34.
 787

19. A. M. Ilyaletdinov, Microbiological transformations, Alma-Ata, Nauka, 1984.

20. P. M. Solozhenkin, V. P. Nebera, I. G. Abdulmanov, Innovations in Mineral and Coal
Processing, Eds. Atak, Onal & Celik, Balkema, Rotterdam, Netherlands, 1998, 495.