Note: Descriptions are shown in the official language in which they were submitted.
117~379
1 B 31748
Effluent treatment
~ his invention relates to effluent treatment and in
particular to the degradation of cyanide containing effluents.
Becau~e of the toxicity of inor g ic cyanides, treat-
ment of cyanide cont~in;ng aqueous effluents is necessary beforethe effluent can safely be discharged into bodies of water such
a rivers, lakes and estuaries.
While cyanide containing effluents can be treated
chemically, e.g. by oxidation with h~pochlorite this tends to be
expen~ive. One microbiological method that has been proposed
involves anaerobic digestion followed by aerobic digestion (see
~SP 3145166)-
It is also known that certain microorganisms, particul-
arly certain fungi, are capable of degrading cyanide to formamide
by means of an enzyme formamide hydrolyase, otherwise termed
cyanide hydratase, (for example see Archives of ~ioohemistry and
Biophysics 151 (1972) pages 468 to 474, and Phytopathology 67 (1977)
pages 1001 to 1006).
We ha~e found that fungal mycelia containing ayanide
hydratase can be immobilised and the resulting immobili~ed
mycelium retains sufficient enzyme activity to be of u~e for treat-
ing oyanide-oontA;n;ng effluents with substantially complete degrad-
ation of the cyanide.
Accordingly we provide a process for the degradation of
inorganic cyanides in effluents comprising contacting the effluent
with an immobilised fungal mycelium containing cyanide hydratase
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for sufficient time to degrade at least 95% of the cyanide ions
in the effluent.
Fungi that may be used include StemPhvlium loti, e.g.
A~CC 11718; ycoleptodiscus terrestris, e.g. C3S 231.53; ~usarium
moniliforme, e.g. ~o. 3104.S~.49a available from the Canadian
Department of Agriculture,Culture Collection, Ottawa, and which
has also been deposited as C~S 161.82; Helm;nthosporium
sor~hicola, otherwise known as Drechslera sor~hicola, eOgO C~S
249.49; Periconia circinata, e.g. C~S 263.37; and Glomerella
tucam nensis, e.g. C3S 132.37. (ATCC ~o. refers to the number
designated by the American ~ype Culture Collection, 12301 Park
Lawn Drive, Rockville, M~wrJla-nd 20852, ~SA, while C~S No. refers
to the number desi-gnated by the Central ~ureau Voor Schi~melcultures,
~aarn, ~etherlands)O Other fungi that may be used and which have
been described in the literature as producing the enzyme include
Collectotrichum graminicola, GloeocercosPora sorghi, ~elminthosPhorium
turcicum, H. maydis, H. carbonum, H. victoriae, and Phoma.
Fungi can be made to grow in two distinct forms, namely
a ball or pellet form or in a mycelial form where the fungal cells
i 20 are diffuse filamentous strands dispersed in the growth medium.It is important that the fungus is grown in the mycelial, as opposed
to ball or pellet, form since, after immobilisation, little dif-
fusion of the effluent can occur into the fungal pellet or ball,
and so a great deal of the active enzyme within the i~mobilised
ball or pellet is not efficiently utilisedO
Often when growing fungi, the ball or pellet form is
obtained: however adjustment of the growth conditions, particularly
- the following parameters:
pl~[,
nitrogen source (nature and amount),
carbon souroe (nature and amount),
amount of phosphorus source,
will enable the mycelial form to be obtained. ~hus with any
particular micro-organism, simple experimentation varying the
above parameters will enable the mycslial form to be obt~;~ed.
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3 ~ 31748
The preferred carbon sources are carbohydrates,
particularly glucose.
When the desired concentration of mycelium has been
- achieved, the enzyme cya ide hydratase may be induced by addingto the culture a low concentration of cyanide ions, e.g. 0.05
to 5, preferably 0.1 to 1 mM per gram of cells (dry wei~ht) and
contin~;ng culturing for 1 to 24, preferably about 12, hours at
20 to 40C.
~he immobilised mycelium may be obtained by flocculat-
- 10 ing the culture with a polyelectrolyte flocculating agent, forexample as described in ~nited K;ngdom Patent Specification
1368650, optionally followed by freezing or drying the flocculated
myceliumO Other methods of i3mobilisation of the mycelium include
entrapment of the harvested mycelium in a suitable material such
as polyacrylamide, an alginate or carrageenan; treatment of the
- mycelium with an adhesive material ~uch as glutaraldehyde before
or after harvesting; and drying the harvested myceliu~, prefer-
ably after treatment of the mycelium with an adhesive material as
described above. ~he immobilised mycelium may be converted into a
suitable particulate form for packing into beds or columns by
extrusion, granulation and/or milling.
When immobilising using polyelectrolyte flocculating
agents, e.g. a mixture of cationic and anionic flocculating agents,
such as 'Primafloc' C7*and 'Primafloc' ~lO*(made by Rhom and Haa3,
Philadelphia), the amount of each flocculating agent employed i9
normally of the order of 4% by weight, based on the cell dry weight.
The use of amounts of flocculating agents in a large excess over
that required to achieve flocculation i9 de~irably avoided, as
~uch large amounts may give relatively tightly bound particles into
which the effluent oannot readily diffuse, thus preventin6 efficient
utilisation of the enzyme in the oells within the particles.
The activity of the flocculated particles can be increased
by dry milling. However the particles should not be milled to too
small a size as then the pressure drop required to cau~e the
effluent to flow through the treatment bsd or column becomes
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uneconomically largeO For this reason the greatest dimension of
the particles is preferably above 1.5 mm.
An immobilisation technique that gives greater activity
involves entrapment of the mycelium in a polyacrylamide (see for
example J. FermentO ~echnol. 53 (1975), 6, p 380-5) or alginate
(see for example ~iotechnol. 3ioeng. 19 (1977) p 387-97). A
particularly suitable technique is to prepare a mixture of the
fungal dispersion and a solution of an alginate, and then to add
this mixture, in dropwise fashion, to a solution, preferably buf-
fered, of a precipitant such as a solution containing calcium ions.Calcium alginate is thus precipitated entrapping the mycelium.
The immobilised mycelium is then packed into beds or
columns wherein it is contacted with the effluentO Cyanide wastes
from chemical plants often are highly alkaline, e.g. having a pE
of at least about lOo For efficient operation the pH is preferably
adjusted to between 7 and 10 before contact with the immobilised
mycelium~
The temperature at which the effluent is treated by the
immobilised mycelium is preferably within the range 0 to 40,
particularly 0 to 30C. Low temperatures, e.g. 4C, tend to
prolong the period over which the enzyme is active.
~ he contact time between the immobilised mycelium and
the effluent will depend on the cyanide content of the effluent,
the desired final cyanide content, and the proportions of immobilised
mycelium to effluent. ~he maximum rate of cyanide ion degradation
will also depend on the proportion of active enzyme in the mycelium.
Generally the maximum rate will lie with;n the range 0.5 to 25 g
cyanide ion per g mycelium per hour.
~n the process of the invention, the effluent treatment
is continued until at least 95%, and in particular at leaat 98%9
of the cyanide have been degraded: the proportion of cyanide ions
that should be degraded will of course depend on the initial, and
desired final, cyanide ion concentration. For discharge into
rivers or other bodies of water, the concentration of cyanide ions
in the discharged effluent is preferably below 10 parts per
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3 31748
million (ppm) by weightO
The immobilised mycelium will require replacement upon
exhaustion of cyanide hydratase activity. ~he replenishment of
the immobilised mycelium may be conducted on a continuous or
intermittent basis. In general, the weight of cyanide ion that
can be treated is over about 200 times the weight of the
immobilised mycelium before exhaustion of the cyanide hydratase
activity.
~ecause of the need to replenish spent immobilised
mycelium, the effluent is preferably treated by passage through
a series of bedR or colu~ns and the columns or beds are replenished
with fresh immobilised mycelium in rotation.
~ or economic operation and in order to achieve the de-
sired residence time a~d acceptable flow rates of effluent, the
bed volume occupied by the immobilised mycelium may be increased
from the volume occupied by the mycelium itself by incorporation
of a~ inert particulate material such as dolomite or silica into
the immobilised mycelium preparation.
The invention is of particular use in treating effluents
from cyanide producing or utilising chemical pl~ntso It is also
of use in treating cyanide containing effluents from an extraction
or metal working plants e.g. electroplating works.
~ he enzyme does not appear to be inhibited by metal ions.
However metals are often complexed with the cyanide. In gener~l
such complexes are not degraded by the enzyme. Some such complexes
are non-toxic and so do not require degradation. Some complexes can
be broken down, e.g. by alkali treatment, and the metal~ recovered,
if desired, by passage through an ion exchange column before or
after degradation of the cyanide by means of the present invention.
The invention is illustrated by the following examples
in which all parts and percentages are expressed by wei~htO
EXAl~IE 1
.`! A mycelium of Stem~hvlium loti (ATCC 11718) was grown
and cyanide hydratase induced by adding a small concentration of
cyanide to the culture and continuing culturing for 2 hoursO lhe
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11'7'~375~
6 ~ 31748
mycelium was immobilised by flocculation, harvesting the floc-
culated mycelium, and drying. 10 mg of the immobilised mycelium
was placed in a shake flask with 100 ml of water containing
2500 ppm of cyanide ions buffered to a pH of about 7.5. After 12
- 5 hours at 28 C the cyanide ion concentration was about 100 ppm.
The aqueous solution was then decanted from the immobilised cells
into a second shake flask to which another 10 mg of the immobilised
mycelium was added. After a further 6 hours at 28C the cyanide
ion concentration was below 10 ppm.
10 EXAMPLæ 2
A cell suspension of Fusarium moniliforme (obtained from
the Canadian Department of Agriculture Culture Collection, Ottawa,
under ~o. 3104.SA.49a) was prepared from an agar slant and
inoculated into 50 ml of medium A and incubated, with sh~k;ng, at
28C for 3 days. 10 ml of the resulting culture was transferred
to 200 ml of medium A in a flask and incubated, with shaking, at
28C for 28 hoursO lhe organism grew in the mycelial form.
Cyanide hydratase was then induced by adjusting the pH
to 7.5 and adding hydrogen cyanide to a concentration of 1 m Molar,
and incubating for 12 hours at 28C.
Medium A, which had a pH of 5.8 - 5.7 had the following
` composition:
glucose 30 g
KH2P04 5 g
casitone 2.1
g 4 2 1 g
Yeast extract 1 g
ECl 0.5
Medium ~ 1 ml
' 30 Distilled water to make up to 1 litre
Medium ~ had the following composition, per litre of
distilled water:
FeS04.7H20 1 g
ZnSO4~7H20 1 g
CuS0405H20 0.15 g
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7 3 31748
MnS04. 4H20 0~1 g
K2MoO4 0.1 g
~Cl (concentrated) suf~icient to just give a
clear solution.
~he m~celium containing the induced cyanide hydratase
was harveated by centrifugation at 10000 g for 10 minutes.
1.75 parts (dry weight) of the mycelium obtained in
Example 2 was incubated at 20C in 1000 parts of an aqueous
solution containing 2.75 parts of potassium cyanide at p~ 8.5.
(~he solution thus contained 1300 ppm of cyanide ion). After
60 minutes incubation, the cyanide ion concentration was below
10 ppm; i.e. over 9~/0 of the cyanide ion had been degraded.
EXAMPIE 4
~he procedure of ~xamples 2 and 3 waæ repeated u8ing
medium C in place of medium A. Medium C had the following
composition:
Molasses 50 g
Phosphate buffer pH 7.2- 20 ml
3~/0 Ammonium Sulphate
aolutio~ 5 ml
40/0 Magnesium Sulphate
solution 0.5 ml
~erric chloride 0.1 ml
Medium ~ 1 ml
Distilled water 975 ml
~ he organism grew in the pelleted form. ~he activity,
i.e. rate of cyanide degradation per unit amount of the organism,
of the enzyme was only about 2~/o of that of the mycelial cells of
Example 3.
EXAMPLE 5
~he procedure of Examples 2 and 3 was repeated u~ing
(i) medium D
(ii) medium
(iii) medium
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8 3 31748
in place of medium A In each case a mixture of mycelial and
pelleted ~rowth was obtained and the enzyme activity was less than
that of the wholly mycelial-form cells of Example 3.
~he compositions of the media D, E and ~, were as follows:
_ ~
glycerol 20 g _
glucose _ 25 g _
~ucrose _ _ 4o g
bacteriolo fi cal peptone lO g _
yeast extract 5 g 3 g
tryptone _ 5 g
co~n steep liquor _ _ 5 g
~ o4 5 g _ 5 ~
MgS04.7H20 _ _ 1 g
Medium ~ _ ~ 2 ~1
Dlstilled waterto l litre to l litreto l litre
:,
EXAMPIE 6
Example 2 was repeated and lO0 part3 of the harvested
; mycelium were suspended in 300 parts of 0.1 M Tris. ECl buffer
(pH 8.5). ~ 2~/o solution of 'Primafloc' C7 (supplied by Rohm and
Haas) at pH 7.0 was added, with continuous stirring, until the
25 cells flocculated. Then an equal amount of a 2% solution of
'Primafloc' AlO (supplied by Rohm and Haas) at pH 6.8 was added.
~he total amount of each polyelectrolyte flocculating a~ent added
was about 4% of the cell dry weight. Ihe flocculated cells were
harvested by filtration, using a Whatman ~o. 1 filter, and then
extruded through a 2 mm aperture. The extruded, wet, immobilised
oells were then cut into partioles of size about 5 mm length and
2 mm diameter.
~XAMPLE 7
30 parts (dry weight) of the extruded, wet, immobilised
cells obtained in Example 6 were incubated at 20C in 1000 parts
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9 3 31748
of an aqueous solution containing 2.75 parts of potassium cyanide
at p~ 8.5. After 54 minutes the cyanide ion concentration was
below 10 ppm. It is seen that, by comparison with Example 3, the
activity of the immobilised enzyme was about 6.5% of the activity
of the cells prior to immobilisation.
EXAMPLE 8
A sample of the extruded, wet, immobilised cells obtained
in Example 6 were dried at 30C for 90 m;nutes. 50 palts of the
dried immobilised cells were tested for enzyme activity as in
Example 7. ~he time taken for the cyanide ion concentration to
reach 10 ppm was about 65 minutes. It is seen that the activity
of the dried cells was about half that of the wet immobilised cells~
~XAMPLE 9
Examples 6 and 7 were repeated, with similar results,
- 15 using a 1% solution of 'Zetagl 94*(supplied by Allied Colloids ~td)
in place of the 2% solution of 'Primafloct C7.
EX~IE 10
Example 9 was repeated using 'Zetag' 64 in place of
'Zetag' 94. Similar results were obtained.
EX~MPIE 11
~xample 6 was repeated and 2 g of the wet, extruded,
immobilised cells were packed into a column 9 mm diameter and 15 cm
; length. A 50 mM aqueous solution of potassium cyanide at pH 8.5
was passed at 20C through the column at a rate of 150 ml hr 10
25 ~he eluent contained 260 ppm of cyanide ions, i.e. about 80yo of
the cyanide ions had been degraded. After 200 hours the eluent
contained about 1200 ppm of cyanide ions.
EXAMPL~ 12
Example 11 was repeated at a flow rate of 50 ml hr 1.
The eluent contained 52 ppm of cyanide ions, i.e. about 96% of
the cyanide ions had been degraded. After 200 hours the eluent
contained 780 ppm of cyanide ions.
Ea~A~I,E 1~
Example 2 was repeated and 100 parts (dry weight) of the
harvested cells were suspended in 1000 parts of Ool M Tris. ECl
* R~g. IM
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~ 31748
; buffer (pE 8.5) at 4C. To the resultant suspension was added
250 parts of acrylamQde, 7 parts of ~,N methylene bisacrylamide,
150 parts of 5% ~ ' tetramethylene di~m;ne and 150 parts of
2.5% ammonium persulphate. The mixture was Lncu~ated at 37C for
5 5 minutes whereupon a gel formed. When the gel had set it was
extruded through a 2 mm aperture and cut into particles as in
~xample 6. The activity of the immobilised enzyme, tested as
described in Example 7~ was abcut 25% of that of the unimmobilised
cellsO
EXAMPlæ 14.
Example 2 was repeated and 5 parts (dry weight) of the
harvested cells were suspended in 1000 parts of 0.1 M TrisO ECl
buffer (pH 8.5). To this suspension was added 20 parts of sodium
alginate. The mixture was stirred for 10 m;nutes and then added
dropwise, with stirring, to 10000 parts of 002 M calcium chloride
made up in ~ris. HCl buffer (pH 8.5). The resultant suspension
of calcium alginate particles, cont~;n;ng the cells, was stirred
for 1 hour and then the particles were harvested by filtration or
a Whatman ~o. 1 filter paper.
The activity of the immobilised cells was assessed as
described in Example 7 a~d found to be about 80Y of that of the
un;mmobilised cells of ~xample 3.
EXAMPIE 15~
A cell suspension of coleptodiscus terrestris
(C3S 231.58) was prepared from an agar slant and inoculated into
50 ml of medium G a~d incubated statically at 28C for 3 daysO
10 ml of the resultant culture was transferred to 2000 ml of
medium H in a flask and incubated, with shaking, at 28C for 28
hours. The organism grew in the mycelial form.
Cyanide hydratase was then induced by adjusting the pE
to 7.5 and adding hydrogen cyanide to a concentration of 1 m Molar
and incubating for 12 hours at 28C. The mycelium was then harvested
by centrifugation at 10000 g for 10 minutes, and th~n immobilised
by the procedure of Bxample 6 and the activity of the immobilised
35 cells a3sessed by the procedure of Example 7. The activity was
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11 ~ 31748
- similar to that of the wet, extruded, immobilised Fusarium moniliforme
cells used in Example 7.
Medium G had the following composition:
V8 tomato and vegetable juice supernatant 200 ml
50 mM phosphate buffer 800 ~1
Medium ~ was the same as medium A except that the amount
of KH2P04 was increased from 5 g to 10 g.
EXAMPLE 16
Example 15 was repeated replacing medium ~ by
10 (i) medium C
(ii) medium D
(iii) medium E
: (iv) medium F
~he organism did not gow in medium C and media D, E and F, gave
pelleted growth.
. EXAMPLE 17
Example 15 was repeated, with similar results, using
Helminthosporium sorghicola (C~S 249.493 in place of coleptodiscus
texrestris and using medium A in place of medium H
'.i'
.
PA/CG~P
23 February 1982
