WO1995015924A1

WO1995015924A1 - Composition and method for sewage treatment using fungal and bacterial enzymes

Info

Publication number: WO1995015924A1
Application number: PCT/US1994/013520
Authority: WO
Inventors: Luis Jimenez; Edward M. Cooney
Original assignee: Reckitt & Colman Inc.
Priority date: 1993-12-09
Filing date: 1994-11-21
Publication date: 1995-06-15
Also published as: SG52242A1; BR9408267A; CA2178344A1; ZA949839B; NZ277630A; AU682565B2; EP0733025A1; AU1293195A; CN1147803A; JPH09509308A

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

COMPOSITION AND METHOD FOR SEWAGE

TREATMENT USING FUNGAL AND BACTERIAL ENZYMES

Field Of The Invention

This invention relates to cellulose degradation by fungal and bacterial enzymes. The combination of enzymes is useful for sewage treatment, particularly in septic tanks.

BACKGROTTND OF THE INVENTION Treatment of sewage with microorganisms is known in the art. Many sewage treatment centers, as well as individual septic tanks, may employ microorganisms for the degradation of sewage. Generally, sewage contains water, organic waste (containing carbohydrates, fats and proteins), and cellulose from paper products. Cellulose may represent up to about 15% of the solids in raw (untreated) sewage.

Typically, the organic, non-cellulosic waste component of sewage is more easily degraded than the cellulose 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 treatment.

The degradation of cellulose to glucose 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 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 cellobiose is hydrolyzed by a beta (1 > 4) glucosidase to glucose. Thus, the breakdown of cellulose to glucose involves a complex of enzymes. Sufficient amounts of these enzy es are not believed to be produced by natural bacteria. The lack of sufficient enzymatic activity is particularly evident in septic tanks, where cellulose sediment is a problem. New methods to reduce the cellulose sediment in raw sewage are needed.

SUMMARY OF THE INVENTION The problems stated above have been solved with the discovery of a sewage treatment composition comprising Bacillus spp. cultures in combination with fungal cellulase. The combination of the extracellular enzymes produced by bacteria cultures from Bacillus spp. and fungal cellulase results in a synergistic degradation of cellulose. Results show a significant enhancement in the production of glucose as a result of 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 enhanced cellulose degradation. Because the composition contains enzymes from naturally occurring microorganisms, it is particularly useful as a septic tank additive.

The invention also provides a novel method for using the Bacillus spp. cultures and fungal cellulase to degrade carbohydrates, protein, fat, and cellulose, and mixtures thereof.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Bacillus spp. are known naturally occurring bacteria as identified on pages 1105 to 1139 of the eighth Edition of Bergey ' ε Manual of Determinative Bacteriology, published by The Williams and Wilkins Co., 1986. Preferred Bacillus species include B. subtiliε, B . licheni formiε , B . mega teri um, and mixtures thereof. More preferably, the bacteria culture is a mixture of B . εubtilis, B . li cheni formiε, and B. megaterium. As known by those skilled in the art, 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 in the composition as spores. The spores 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 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 frequency 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 for a composition prepared as a typical septic tank additive preferably employs at least about 10^ spores/g of composition (active ingredient) , with the upper limit concentration of spores generally limited only by cost. More preferably at least 10^ spores/g and most preferably from 10^ to 10^ spores/g of composition (active ingredient) is employed in the composition.

The cellulase is isolated from Aεpergilluε niger fungus. The enzyme may be extracted from the fungal culture by any known means, and is widely available commercially from, for example, Novo Nordisk, Ct.; Sigma Chemical, St. Louis, Mo.; and George A.

Jeffrey's 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 employed in this invention. The Bacillus spp. bacteria 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 high 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 preferably from 1500 to 2500 CU/g and most preferably from 1500 to 2000 CU/g of active ingredient portion of composition. The ratio of bacteria culture to fungal 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 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, NaS04, CaCθ3, mixtures thereof and so on), inert preparations (such as milorganite) , mixtures thereof, and so on. The composition may be prepared as a liquid or powder by any means known to those skilled in the art.

As previously described, the enzymes utilized in the inventive composition are produced by naturally occurring organisms. Thus the composition is useful for many industrial applications where broad based degradation of components typical of sewage (e.g. carbohydrates, proteins, fats and cellulose) is desired.

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 combination was found to be more effective in degrading cellulose than when a larger amount of plain fungal enzyme was used. The inventive combination offers a broad based sewage treatment system as well as a means of producing glucose from cellulose, particularly useful in industrial applications where cellulose is a waste product.

The enzymatic action of the inventive composition may occur over a wide pH range. Optimally, the pH range of the media to be treated falls within about 4 to about 10, with more preferably the pH having a value between 6 and 8. The temperature range of the media to be treated may vary greatly, although optimum enzymatic action preferably occurs within a temperature range of from about 10°C to about 45°C and more preferably between 20°C and 35°C. 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 fungus is an aerobic microorganism and the Bacillus spp. a facultative anaerobic microorganism, thus the oxygen content of the substrate environment must be considered in preparing the composition.

As known to those skilled in the art, the dosage, frequency of use, as well as the concentration of the active ingredient portion of the composition are interdependent variables that will also vary widely depending upon the environment to be treated, the concentration of particles to be degraded, prior usage of microorganisms, and so on. Adjustments to these 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 the composition is at least about 10 g, more preferably at least about 100 g (with the upper limit of the amount used limited primarily by cost), and most preferably from 150 g to 1000 g.

The invention is further illustrated, but not limited to, the following examples.

EXAMPLES Cellulose degradation was measured by glucose production, as determined by the Dinitrosalicylic acid procedure (DNS), as described in Aibba, S., K. Kitai, and T. Imanaka, Appli ed Environmental Mi crobiology, Vol. 46, pp. 1059-1065 (1983) .

The compositions described in the examples used spores isolated from Bacill us subtiliε , Bacilluε licheniformiε, and Bacillus mega terium, and fungal cellulase isolated from Aεpergilluε niger. Both the spores and cellulase were obtained from the George A. Jeffreys Company. The culture has a count of 10° spores/gram of active ingredient portion of the composition. The cellulase had a specific activity of 1600 CU/g of active ingredient portion of the composition. (As obtained from supplier, actual cellulase enzyme activity was approximately 128,000 CU/g.) The milorganite was purchased from Milwaukee Metropolitan Sewage District, Milwaukee, WI.

Examples 1 and 2 and Comparative Examples 1 and 2 Synthetic sewage was prepared with 5% protein, 5% fat, 5% cellulose, and the remainder distilled water. The synthetic waste was placed in a 35 ml test tube for each composition tested.

INVENTIVE COMPOSITION A

Inqredients Weight Actual Percentage Amounts

Bacilluε spp. 40% 200 g. Spores

NaCl 20% 100 g

NaS0₄ 15% 75 g

CaCOβ 24.5% 122.5 g

Cellulase 0.5% 2.5 g (640 CU/g)

Total Volume 1Q0%. 500 q.

Example 1 Composition A was diluted 10% with the waste for a final cellulase concentration of 0.050% (64 CU/g) . After 48 h glucose production (thus indicating the level of cellulose degradation) was measured as 4.83 g/lt using the DNS method, as recorded in Table I below.

Example 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. Comoarative Example 1 Example 1 was repeated with the exception that only cellulase was added to the synthetic waste at a concentration of 0.5% (640 CU/g). After 48 h glucose production was measured by DNS as 1.41 g/lt, as recorded in Table I.

Comparative Example 2 Example 1 was repeated with the exception that Composition A did not have any fungal cellulase and CaCθ3 and NaSθ4 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 Examples 1 and 2 and Comparative Examples 1 and 2. When the 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 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 both bacterial and fungal enzymes. Similar results were found using raw sewage (Table 2).

Tabl e 1

Glucose production in synthetic waste

Glucose (g/lt)

Example 1 4.83

Example 2 1.31

Comparative Example 1 1.41

Comparative Example 2 0.17

Inventive Composition B

Ingredients Weight Percentage Actual Amounts

Bacilluε spp. 40% 200 g Spores

NaCl 20% 100 g

NaSθ 15% 75 g

CaC03 24.5% 122.5 g

Cellulase 0.5% 2.5 g (640 CU/g)

Total Volume 100% 500 g

Example 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 level of cellulose degradation) was measured as 0.291 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 Composition B or the enzyme by itself. As recorded in Table 2, no glucose was detected when samples were analyzed by the DNS procedure.

Comparative Example 3 Example 3 was repeated with the exception 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.

Comparative Example 4 Example 3 was repeated with the exception that Composition B did not have any fungal cellulase and CaCθ3 and NaS04 were replaced with milorganite. After 48 h glucose production was measured by DNS as 0.029 g/lt, as recorded in Table 2.

Table 2 Glucose production of raw sewage

Glucose (g/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 invention.

Claims

THAT WHICH IS CLAIMED IS:

1. A composition comprising Bacillus spp. 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 subtiliε, Bacillus licheniformiε , 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 Aεpergilluε 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 filler is an alkali metal salt selected from the group consisting of NaCl, NaSO, CaC03 and mixtures thereof.

8. A composition according to claim 1 wherein said cellulase is isolated from Aεpergilluε niger fungus.

9. A method of using a composition comprising Bacilluε 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 licheniformiε, 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 Aεpergilluε 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 licheniformiε, and Bacillus megaterium.

16. A method according to claim 15 wherein said fungal cellulase is isolated from Aεpergilluε 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 licheniformiε, Bacilluε megaterium, and mixtures thereof.

19. A composition according to claim 18 wherein said spores obtained are a mixture of said Bacilluε spp.

20. A composition according to claim 19 wherein said cellulase is isolated from Aεpergilluε 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 selected from the group consisting of NaCl, NaS04, CaCθ3. and mixtures thereof.