Note: Descriptions are shown in the official language in which they were submitted.
~WO 95115924 2 1 7 8 3 4 4 PCT/IJS94/13520
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~ MPOgITION AND M~T~IOD POR SEWAGE
TpT.'A'r'MF'NT ~JSING FIJN('.Ar. AN,D B~cTT~T~TAT~ T'N7Y~rl~q
Field Of The Invention
This invention relates to cellulose
5 degradation by fungal and bacterial enzymes. The
com.bination of enzymes i5 useful for sewage treatment,
particularly in septic tanks.
EACKGROUND OF T~T~ I~VE~TION
Treatment of sewage with microorganisms is
10 known in the art. Many sewage treatment centers, as
well as individual septic tanks, may employ
micronrg~n; F:mq for the degradation of sewage.
Generally, sewage contains water, organic waste
(containing carbohydrates, fats and proteins), and
lS cellulose from paper products. Cellulose may represent
up to about 15% of the solids in raw ~untreated)
sewage .
Typically, the organic, non-cellulosic waste
., ^nt of sewage is more easily degraded than the
20 c.o~ se component. Carbohydrates, fats and proteins
making up the organic waste are fairly easily digested
by extracellular enzymes released outside the cell of
selected bacteria. The degradation of cellulose,
however, remains a problem in many forms of sewage
25 treatment.
The degradation of cellulose to ~lucose is a
stepwise process. First, cellulose is hydrolyzed by
the action of an endoglucanase that breaks bonds along
the amorphous regions of cellulose. This enzymatic
3 0 reaction carries out the cleavage of the beta ( 1 > 4 )
bonds producing cellobiose which will be removed from
the nonreducing ends of the molecule by the action of a
beta (1 > 4) exoglucanase. After this, the c-~llo~iose
is hydrolyzed by a beta (1 > 4) glucosidase to glucose.
35 Thus, the breakdown of cellulose to glucose involves a
complex of enzymes. Sufficient amounts of these
2 ~ 78344
WO 95/15924 -, ~ PCT/US94/13520
--2--
enzymes are not believed to be produced by natural
bacteria. The lack of sufficient enzymatic activity is
p~rticularly evident in septlc tanks, where cellulose
sediment is a problem. New methods to reduce the
5 cellulose sediment in raw sewage are needed.
srT~RY OF Tl Tr' INVE~TION
The problems stated above have been solved
with the discovery of a sewage treatment composition
comprising Bacillus 8pp. cultures in combination with
10 fungal cellulase. The combination of the extracellular
enzymes produced by bacteria cultures from Bacillus
spp. and fungal c~ lRce results in a synergistic
degradation of cellulose . Results show a si~rnif icant
~nh~n~ t in the production of glucose as a result of
15 cellulose degradation when sewage containing cellulose
is contacted with the inventive composition. The
composition is a broad based system capable of breaking
down carbohydrates, fats and proteins in addition to
~nh;ln~ ~(1 cellulose degradation. secause the
20 composition contains enzymes from naturally occurring
mi~L.uLyC~nisms~ it is particularly useful as a septic
tank additive.
The invention also provides a novel method
for using the Bacillus spp. cultures and fungal
25 cellulase to degrade carbohydrates, protein, fat, and
cellulose, and mixtures thereof.
DET~TL~n DEst~RTPTION OF Tr-rr~ ) EMr~nDIMF~T
Bacillus spp. are known naturally occurring
bacteria as identified on pages 1105 to 1139 of the
30 eighth Edition of Bergey~s Nanual of Determinative
Bacteriolo~y, published by The Williams and Wilkins
Co., 1986. Preferred E~acillus species include B.
subtilis, B. licheniformis, B. megaterium, and mixtures
thereof. ~5ore prefera~ly, the bacteria culture is a
35 mixture of B. subtilis, E~, licheniformis, and B.
megaterium. As known by those skilled in the art,
21 783~4 ~ s 9~; / 13 520
;Jj 06 Ji)L
bacteria cultures may be prepared as spores to extend
the period that the cultures may be stored. Preferably
for convenience of storage, the Bacillus spp. cultures
are present iIl the composition as spores. The spores
5 become enzyme producing organisms when exposed to
nutrients such as sewage. When exposed to sewage the
spores generate into bacteria producing extracellular
enzymes that are particularly effective in degrading
carbohydrates, fats and proteins. The spore count of
10 Bacillus spp. employed in the composition may vary
greatly, depending upon the type of sewage to be
treated, the size of the sewage treatment facility, the
fre~uency of treatment of the sewage with the
composition, and so on.
As used herein, the active ingredient portion
of the composition is defined as the bacteria culture
and fungal enzyme. A concentration range of bacteria
cultures or a composition prepared as a typical septic
tank additive preferably employs at least about 104
20 spores/g of composition (active ingredient), with the
upper limit concentration of spores generally limited
only by cost. More preferably at least 106 spores/g
and most preferably from 1Ob to 108 spores/g of
composition (active ingredient~ is employed in the
25 composition.
The cellulase is isolated from Aspergillus
niger fungus. The enzyme may be extracted from the
fungal culture by any known means, and is widely
available comnercially from, for example, Novo Nordisk,
30 Ct.; Sigma Chemical, St. Louis, Mo.; and George A.
Jeffreys Company, Salem, Va. Because the fungus is
aerobic, and sewage treatment is largely in a submerged
anaerobic environment, it is preferred that the
cellulase enzyme is separated from the fungus as
35 employed in this invention. The Bacillus spp. bacteria
; . v ,,
21 783~4 ~ J~J, / 13 520
Ti~f~;v 06 JUl'95
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are facultative anaerobic and thus thrive in the
typically anaerobic conditions of sewage treatment.
As with the spore count of the bacteria
cultures, the specific activity and amount of the
fungal cellulase enzyme employed in the composition is
widely variable and may be adjusted according to the
enzymatic needs of the system employing the
composition. For example, with waste systems having a
particularly hiç~h content of cellulose, large amounts
of the cellulosic enzymes would be preferred. For use
as a septic tank additive, the activity of the fungal
enzyme emPloyed is preferably at least about 1000 CU/g
of active ingredient portion of composition (with the
upper limit of concentration of enzyme generally
limited only by cost ) . For reasons of economy in
formulating septic tank additives the enzyme range is
more pre f erably f rom 15 0 0 to 2 5 0 0 CU/ g and mos t
preferably from 1500 to 2000 CU/g of active ingredient
portion of compos i t ion .
The ratio of bacteria culture to ~ungal
enzyme may vary greatly. Preferably the ratio is
anywhere between about 10:90 to about 99.99:0.01
percent by weight of active ingredient bacteria
culture: fungal enzyme. As known to those skilled in
_ 25 the art, the ratio may be adjusted depending upon the
type of material to be treated, the spore count and
specific activity of raw materials, and so on.
The composition may also include optional
fillers and additives to facilitate storage or delivery
of the spores and fungal enzyme into the treatment
facility. Fillers that may be used include, but are by
no means limited to, alkali metal salts (such as NaCl,
Na2SO4, CaCO3, mixtures thereof and so on), inert
preparations (such as milorganite), mixt~res thereof,
3 5 and so on .
S~iE-T
~Wo s~/15924 ~ 2 t 7 8 3 4 I PCTIUSg4/13520
The composition may be prepared as a Iiqui~
or powder by any means known to those skilled in the
art .
As previously described, the enzymes utilized
5 in the inventive composition are produced by naturally
occurring organisms. Thus the composition is useful
for many industrial applications where broad based
degradation of r^~r~nPnts typical of sewage (e.g.
carbohydrates, proteins, fats and cellulose) is
l 0 des i red .
As shown in the Examples section hereinafter,
the combination of Bacillus spp. enzymes and the fungal
cellulase has been found to be synergistic. A smaller
amount of bacteria and fungal enzymes used in
15 ' ` in~tlon was found to be more effective in degrading
cellulose than when a larger amount of plain fungal
enzyme was used. The inventive combination of fers a
broad based sewage treatment system as well as a means
of p~oducing glucose from cellulose, particularly
20 useful in industrial applications where cellulose is a
waste product.
The enzymatic action of the inventive
composition may occur over a wide pX range. Optimally,
the pH r~nge of the media to be treated falls within
25 about 4 to about l0, with more preferably the pH having
a value between 6 and 8. The temperatu~e range of the
media to be treated may vary greatly, although optimum
enzymatic action preferably occurs within a temperature
range of from about 10C to about 45CC and more
30 preferably between 20C and 35C. Degradation of
cellulose may also occur with enzymes separated from
the Bacillus spp. and combined with cellulase of a
fungal origin (separated or unseparated from the
fungus). As known to one skilled in the art, the
35 fungus is an aerobic microorganism and the Bacillus
spp. a facultative anaerobic microorganism, thus the
.
wo 95/l592~ 2 ~ 7 8 3 4 4 PCT/I~S94/13520
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oxygen content of the substrate e~vironment must be
considered in preparing the composition.
As known to those skilled in the art, the
dosage, fre~uency of use, as well as the ct~nr~ntration
5 of the active ingredient portion of the composition are
interdependent variables that will also vary widely
depending upon the environment to be treated, the
c~n-~Pntration of particles to be degraded, prior usage
of microorganisms, and so on. Adjustments to these
10 variables may be accomplished by routine procedures
known to those skilled in the art. For example, for
use as a septic tank additive (with the septic tank
typically having a capacity of about 1000 gallons), an
effective amount of the active ingredient portion of
15 the composition is at least=about 10 g, more preferably
at least about lO0 g (with the upper limit of the
amount used limited primariIy by cost), and most
preferably from 150 g to 1000 g.
The invention is further illustrated, but not
20 limited to, the following examples.
MPT.l;'C
Cellulose degradation was measured by glucose
production, as determined by the Dinitrosalicylic acid
procedure (DNS), as described in Aibba, S., K. Kitai,
25 and T. Imanaka, Applied Environmental l~icrobiolos~y,
Vol. 46, pp. 1059-1065 (1983).
The compositions described in the examples
used spores isolated from ~acillus subtilis, Ri~Ci 7 7~q
licheniformis, and ~3acil l us megaterium, and fungal
30 cellulase isolated from Aspergillus niger. Both the
spores and cellulase were obtained from the George A.
.Jeffreys Company. The culture has a count of 108
spores/gram o~ active ingredient portion of the
composition . The cellulase had a specif ic activity of
35 1600 CUJg of active ingredient portion of the
composition. (As obtained -from supplier, actual
~WO 95/15924 2 ~ 7 8 3 4 4 PCr/Ussd~113520
cellulase enzyme activity was approximately 128, 000
CU/g. ) The milorganite was purchased from M; l~-allk~e
Metropolitan Sewage District, Milwaukee, WI.
EY~mnleR l and 2 and r -rative F!Y~mnl DR 1 and 2
Synthetic sewage was prepared with 596
protein, 5% fat, 596 cellulose, and the r~mA;nf9~r
distilled water. The synthetic waste was placed in a
35 ml test tube for each composition tested.
2 ~ 7 8 3 4 4 ~ ,, ,i ~ ~ ,i 4 / 1 3 5 2 0
-8- ~s~ ; 06JUL 93
lNV~LlV~ MposITIoN A
Inore~i ents
Percenta~e Amounts
3acillus spp. 40% 200 g
Spores
NaCl 20% 100 g
Na2S4 15% 75 g
CaCO3 24 . 5% 122 . 5 g
Cellulase 0 . 5% 2 . 5 g
~640 CU/g~
Total Volume LQQ~
Exiq le 1
Composition A was diluted 1~% with the waste
for a final cellulase concentration of 0 . 050% (64
-- CU~g). After 48 h glucose production ~thus indicating
10 the level of cellulose degradation) was measured as
4 . 83 g/lt usin~ the DNS method, as r~cor~ed in Table I
below .
E~riqmnle 2
Example 1 was repeated with the exception
that Composition A was diluted 2 . 5% with waste for a
final cellulase concentration of 0.0125~ (16 CU/g).
After 47 h glucose production was measured by DNS as
1. 31 g/lt, as recorded in Table I below.
~ ~r; .i: ~r, Sht~T
2 t 7 8 3 4 4 ~ 3 5 2 0
.J~;~ O ~ JUL '
g
C~mnar2tive Ex~mnle 1
Example 1 was repeated with the exception
that only cellulase was added to the synthetic waste at
a concentration of 0.596 (640 CU/g~ . After ~8 h glucose
5 production was measured by DNS as 1. 41 g~lt, as
recorded in Table I.
C omoa ra t i ve Ex;~ mr~ 1 e 2
Example 1 was repeated with the exception
that Composition A did not have any fungal cellulase
10 and CaCO3 and Na2SO4 were replaced with an equivalent
amount milorganite. After 48 h glucose production was
measured by DNS as 0.17 g/lt, as recorded in Table I.
Table 1 summarizes data obtained in Ex2mples
_' 1 and 2 and Comparative Examples 1 and 2. When the
15 fungal cellulase was added to the culture a significant
increase in the production of glucose was detected. As
Examples 1 and 2 show more or similar amount of glucose
was produced by the composition, once the cellulase was
added, than with the enzyme by itself though the
20 cellulase concentration in Examples 1 and 2 was
approximately ten times less than with the enzyme by
itself. Therefore, these major differences between the
enzymes by itself and the combination of fungal and
bacterial enzymes were due to the synergistic effect of
25 both bacterial and fungal enzymes. Similar results
were found using raw sewage (Table 2).
A,';,~, ;L~D SHEET
21 78344 ~ : ; / 1 3 520
o~ ~UL`'95
-10-
Ta~le I
Glucose l~roduction in svnthetic w~ste
Glucose (c/lt~
Example 1 4. 83
Example 2 1. 31
Comparative Example 1 l . 41
Comparative Example 2 0.17
InventiYe CO~osition ~
~,In~re~i ents Weicrht Percenta~e Actual Amo~nts
Bacillus spp. 40% 200 g
Spores
NaCl 20% 100 g
Na2So4 15% 75 g
CaCO3 24.5% 122.5 g
Cellulase 0 . 5% 2 . 5 g
( 640 CU/g)
Total Volume 100% 500 g
F~mn l e 3
Raw sewage (obtained from the Ridgewood waste
water treatment plant, Ridgewood, N.J. ) was placed into
a 35 ml tube and Composition B was diluted 2 . 5~ for a
final cellulase concentration of 0 . 0125% (16 CU/g) .
After 48 h glucose production (thus indicating the
15 level of cellulose degradationJ was measured as 0.291
A;,~ ,LD S~ T
2 1 783~4 ~ f ~ 3
~P~ 06 JUl'9
-11 -
g/lt using the DNS method, as recorded in Table 2
below .
A Control was also run, where glucose
production was measured without the presence of
5 Composition B or the enzyme by itself. As recorded in
Table 2, no glucose was detected when samples were
analyzed by the DNS procedure.
Com~arative FxAmnle 3
Example 3 was repeated with the exception
10 that only cellulase was added to the sewage at a
concentration of 0.5% ~640 CU/g). After 48 h glucose
production was measured by DNS as 0.128 g/lt, as
~~ recorded in Table 2.
Co-narative Ex;~rnle 4
Example 3 was repeated with the exception
that Composition B did not have any fungal cellulase
and CaCO3 andNa2SO4 were replaced with milorganite.
After ~8 h glucose production was measured by DNS as
0 . 029 g/lt, as recorded in Table 2 .
TabLe 2
Glucose ~roduction of raw sewaae
Glucose (c~lt)
Example 3 0 . 291
Control 0 . 000
Comparative Example 3 0.128
Comparative Example 4 0.029
The invention has been described above with
particular reference to preferred embodiments. A
skilled practitioner familiar with the above-detailed
description can make many modifications and
substitutions without departing from the scope and
spirit of the irlvention.
~.i,'~'..;., ~11~'Er
