CA2178344A1 - Composition and method for sewage treatment using fungal and bacterial enzymes - Google Patents
Composition and method for sewage treatment using fungal and bacterial enzymesInfo
- Publication number
- CA2178344A1 CA2178344A1 CA002178344A CA2178344A CA2178344A1 CA 2178344 A1 CA2178344 A1 CA 2178344A1 CA 002178344 A CA002178344 A CA 002178344A CA 2178344 A CA2178344 A CA 2178344A CA 2178344 A1 CA2178344 A1 CA 2178344A1
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- Prior art keywords
- bacillus
- composition
- cellulase
- cultures
- composition according
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-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biodiversity & Conservation Biology (AREA)
- Water Supply & Treatment (AREA)
- Environmental & Geological Engineering (AREA)
- Hydrology & Water Resources (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Treatment Of Sludge (AREA)
- Detergent Compositions (AREA)
Abstract
Extracellular enzymes from Bacillus spp. cultures in combination with fungal cellulase have been found to synergistically degrade cellulose. The composition is also useful in degrading carbohydrates, fats and proteins thus lending its usefulness to sewage treatment.
Description
~WO 95115924 2 1 7 8 3 4 4 PCT/IJS94/13520 ~ 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 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 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 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 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 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
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 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 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 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 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 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
Claims (24)
1. A composition comprising Bacillus spp.
cultures and fungal cellulase.
cultures and fungal cellulase.
2. A composition according to claim 1 wherein said bacterial culture is in a spore form.
3. A composition according to claim 2 wherein said spores are obtained from Bacillus spp.
cultures selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, and mixtures thereof.
cultures selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, and mixtures thereof.
4. A composition according to claim 3 wherein said spores obtained are a mixture of said Bacillus spp.
5. A composition according to claim 4 wherein said cellulase is isolated from Aspergillus niger fungus.
6. A composition according to claim 5 further comprising a filler selected from alkali metal salts and inert metal preparations.
7. A composition according to claim 6 wherein said filter is an alkali metal salt or an alkaline earth metal salt selected from the group consisting of NaCl, Na2So4, CaCO3 and mixtures thereof.
8. A composition according to claim 1 wherein said cellulase is isolated from Aspergillus niger fungus.
9. A method of using a composition comprising Bacillus spp. cultures in combination with fungal cellulase to degrade sewage comprising carbohydrates, proteins, fats, cellulose, or mixtures thereof.
10. A method according to claim 9 wherein said cultures are present in a spore form prior to contacting said composition with said sewage.
11. A method according to claim 10 wherein said cultures are selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, and mixture thereof.
12. A method according to claim 11 wherein said culture is a mixture of said Bacillus spp.
13. A method according to claim 9 wherein said cellulase is isolated from Aspergillus niger fungus.
14. A method of producing glucose from cellulose employing fungal cellulase and extracellular enzymes produced by Bacillus spp. cultures.
15. A method according to claim 14 wherein said Bacillus cultures are a combination of Bacillus subtilis, Bacillus licheniformis, and Bacillus megaterium.
16. A method according to claim 15 wherein said fungal cellulase is isolated from Aspergillus niger fungus.
17. A method to degrade sewage comprising contacting said sewage with an effective amount of a composition comprising Bacillus spp. spores and fungal cellulase sufficient to degrade carbohydrates, proteins, fats, cellulose, and mixtures thereof.
18. A septic tank additive comprising fungal cellulase and spores obtained from Bacillus spp.
cultures selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, and mixtures thereof.
cultures selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, and mixtures thereof.
19. A composition according to claim 18 wherein said spores obtained are a mixture of said Bacillus spp.
20. A composition according to claim 19 wherein said cellulase is isolated from Aspergillus niger fungus.
21 A composition according to claim 20 further comprising a filler selected from alkali metal salts and inert metal preparations.
22. A composition according to claim 21 wherein said filler is an alkali metal salt or an alkaline earth metal salt selected from the group consisting of NaCl, Na2SO4, CaCO3 and mixtures thereof.
23. A method according to claim 13 in which the composition further comprises a filler selected from the group consisting of alkali metal salts, alkaline earth metal salts and inert metal preparations.
24. A method according to claim 23 in which the filler is an alkali metal salt or an alkaline earth metal salt selected from the group consisting of NaCl, Na2SO4, CaCO3 and mixtures thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US16460993A | 1993-12-09 | 1993-12-09 | |
| US08/164,609 | 1993-12-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2178344A1 true CA2178344A1 (en) | 1995-06-15 |
Family
ID=22595281
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002178344A Abandoned CA2178344A1 (en) | 1993-12-09 | 1994-11-21 | Composition and method for sewage treatment using fungal and bacterial enzymes |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0733025A1 (en) |
| JP (1) | JPH09509308A (en) |
| CN (1) | CN1147803A (en) |
| AU (1) | AU682565B2 (en) |
| BR (1) | BR9408267A (en) |
| CA (1) | CA2178344A1 (en) |
| NZ (1) | NZ277630A (en) |
| SG (1) | SG52242A1 (en) |
| WO (1) | WO1995015924A1 (en) |
| ZA (1) | ZA949839B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU5993096A (en) * | 1996-05-21 | 1997-12-09 | Bevil S.P.R.L. | Biological purification of septic tanks by extender effect |
| US5875229A (en) * | 1996-10-15 | 1999-02-23 | Motorola Inc. | System and device for, and method of, detecting, characterizing, and mitigating deterministic distortion in a communications network |
| US5812602A (en) * | 1996-11-14 | 1998-09-22 | Motorola Inc. | System and device for, and method of, communicating according to a trellis code of baseband signals chosen from a fixed set of baseband signal points |
| US5862179A (en) * | 1997-02-14 | 1999-01-19 | General Datacomm, Inc. | Mapper for high data rate signalling |
| US5822371A (en) * | 1997-02-14 | 1998-10-13 | General Datacomm Inc. | Mapper for high data rate signalling |
| US5838724A (en) * | 1997-02-14 | 1998-11-17 | General Datacomm, Inc. | Spectral and power shaping mapper for high data rate signalling |
| US6185249B1 (en) * | 1999-01-28 | 2001-02-06 | Ic Tel Inc. | Translation table design for a PCM modem |
| US6560277B2 (en) | 2001-02-09 | 2003-05-06 | Pc Tel, Inc. | Distinguishing between final coding of received signals in a PCM modem |
| EP2319504A1 (en) | 2009-11-07 | 2011-05-11 | Laboratorios Del. Dr. Esteve, S.A. | Pharmaceutical solid dosage form |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2482130A1 (en) * | 1980-05-12 | 1981-11-13 | Lumer Ste Indle Produits Chimi | Inoculum for septic tanks and purificn. digesters - prepd. by drying bacterial cultures with molecular sieve, then adding enzyme and dispersant |
| AU8496382A (en) * | 1981-07-16 | 1983-01-20 | Unisearch Limited | Enzymic process to hydrolyze celluloses |
| US4940539A (en) * | 1989-05-08 | 1990-07-10 | Semco Laboratories, Inc. | Grease trap construction |
| JP3361095B2 (en) * | 1991-08-30 | 2003-01-07 | ユナイテッド ラボラトリーズ インコーポレイテッド | Separation method of lipophilic hydrophobic substance in washing water |
| FR2699525B1 (en) * | 1992-12-22 | 1995-03-24 | Hecke Jean Claude Van Den | Composition of matter for the chemical, physical and biological purification of polluted aqueous liquids, its preparation process and purification process using this composition. |
-
1994
- 1994-11-21 NZ NZ277630A patent/NZ277630A/en unknown
- 1994-11-21 WO PCT/US1994/013520 patent/WO1995015924A1/en not_active Application Discontinuation
- 1994-11-21 JP JP7516207A patent/JPH09509308A/en active Pending
- 1994-11-21 AU AU12931/95A patent/AU682565B2/en not_active Ceased
- 1994-11-21 SG SG1996001168A patent/SG52242A1/en unknown
- 1994-11-21 EP EP95904120A patent/EP0733025A1/en not_active Withdrawn
- 1994-11-21 CA CA002178344A patent/CA2178344A1/en not_active Abandoned
- 1994-11-21 CN CN94194795A patent/CN1147803A/en active Pending
- 1994-11-21 BR BR9408267A patent/BR9408267A/en not_active Application Discontinuation
- 1994-12-09 ZA ZA949839A patent/ZA949839B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| AU1293195A (en) | 1995-06-27 |
| CN1147803A (en) | 1997-04-16 |
| SG52242A1 (en) | 1998-09-28 |
| WO1995015924A1 (en) | 1995-06-15 |
| ZA949839B (en) | 1996-06-10 |
| AU682565B2 (en) | 1997-10-09 |
| JPH09509308A (en) | 1997-09-22 |
| NZ277630A (en) | 1997-10-24 |
| BR9408267A (en) | 1996-12-10 |
| EP0733025A1 (en) | 1996-09-25 |
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