CA1041681A - Method for the purification of waste water - Google Patents

Abstract

METHOD FOR THE PURIFICATION OF WASTE WATER

ABSTRACT OF THE DISCLOSURE
Effluent is removed from water by treating the effluent containing water with an enzyme of a microorganism having an aqueous ambience and/or an enzyme of the type employed in detergent preparations. Previous analytic studies of the ambience of aqueous environments have shown the presence of enzymes therein. However, it was heretofore unknown that any of these enzymes could be employed for purifying water. Micro-organisms which have an aqueous ambience are generally a source of the enzymes within the scope of this invention. Preferably, microorganisms whose aqueous ambience is polluted are employed for obtaining the enzymes, and particularly preferred enzymes are derived from microorganisms which thrive in the water which is to be purified. A particularly suitable enzyme is, or instance, obtained from the microorganisms which are employed in conventional activated sludge and trickling filter methods to biologically purify water. Quite unexpectedly it has been found that enzymes produced by these microorganisms, rather than the microorganisms themselves, may be employed to purify effluent containing water. The enzyme producing microorganisms within the scope of this invention are not purestrains; they are mixed populations, such as usually occur in bodies of water, particularly in effluent containing water.

Description

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1041681 1 ~
¦ METHOD FOR THE PURIFIGATION OF WASTE WATER ~
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BACKGROUND OF THE INVENTION
This invention relates to a method for purifying efluent ~
containing water, and more particularly to a method for bio- ;~ ~ -¦ ~hemically processing effluent containing water for effluent ¦
removal therefrom. In general, organic effluent in water is biologically decomposed by treatment with bacteria and certain types of plants. Conventionally~ chemically active aerobic bacteria have been employed to effect organic effluent de- 1 composition. If necessary, the effluent containing water has b en aerated to activate the bacteriologic mechanism for ^`
~ dacomposition.
¦ In these prior processes it was generally found necessary to ~employ trickling filters or activated sludge to biochemically ¦
purify the effluent containing water. Both of these filtration ~ techniques require substantial expenditures for equipment and i nonrecoverable raw material, and the expenses for these .
techniques escalate proportionately to the volume of water processed.
A disadvantage of these prior methods is the large quantity `~
of microorganic sludge they produce. Because of its ma~eup, this sludge LS difXicult to dispose of. Another disadvantage of theseprior methods is their inability to satisfactorily process highly variable quantities of water. To treat occasional '~ ' !.,.. ' : , ' . i li i ~
¦ ! l heavy flows or effluent containing water without exceeding the capacity of the processing system, it is usual to design the system on a larger scale than would be required to process normal or usual volumes of effluent containing waterO ¦
Therefore~ the instant invention provides an improved method for biologically treating effluent containing water which is highly effective, less costly than prior methods, ;;
which produces less microorganic sludge than prior methods, and which is capable of processing sudden peak loads of effluent, without special provision being made therefor.
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SUMMARY OF THE INVENTION
Generally speaking, according to the instant invention~
effluent is removed from water by treating ~he effluent con-taining water with an enzyme of a microorganism having an aqueous ambience and/or an enzyme of the type employed in detergent prepara~ions.
Previous analytic studies of thé ambience of aqueous ; 1~ 1 environments have shown the presence of enzymes therein. 1 However, it was heretofore unknown that any of these enzymes could be employed for purifying water. -Microorganisms which have an aqueous ambience are generally a source of the enzymes within the scope of this invention. ~ -Preferably,microorganisms whose aqueous ambience is polluted `~ ;~
are employed for obtaining the enzymes, and particularly : 1, ' ,J,. A, . , , . ' .
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preferred enzymes are derived from microorganisms which thrive I ` ~
in the water which is to be purified. A particularly suitable ¦ ~ `
enzyme is, for instance, obtained from the microorganisms which are employed in conventional activated sludge and trickling filter methods to biologically purify water. Quite f unexpectedly it has beea found that enzymes produced by these I
microorganisms, rather than the microorganisms themselves, may be employed to purify effluent containing water.
The enzyme producing microorganisms within the scope of this invention are not pure strains; they are mixed populations, such as usually occur in bodies of water, particularly in effluent containing water.
Accordingly, it is an object of this invention to provide 1 7 a method for purifying effluent containing water which lncreases the capacity of a puri~ication plant withou~ a concomitant increase in the size of the plant, per se.
¦ Another object of the invention is to provide a method ~ for purifying effluent containing water which is compatible ~
;1~ with prior conventinnal methods and synergistically improves ~ ~- the effectiveness of any conventional process employed in conjunction therewlth. -I A further object of the invention is to provide a method for pu~ifying effluent contalnlng ~ater thlch lncludes treatlng ',,' ~ , ' ~;`.

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1041~81 the effluent containing water with an enzymatic preparation.
Still another object of the invention is to purify effluent containing water by adding thereto enzymes of microorganisms :~
which inhabit the ambience of the effluent containing water under treatment. :: ~
Another object of the inven~ion is to provide a method :
for purifying water which includes culturing microorganisms ¦ `
employed in conventional biochemical purification processes .
for obtaining enzymes therefrom and treating the effluent containing water with the extracted enzymes~
. Still other objects and advantages of the invention will . :
in part be obvious and will in part be apparent from the specification. .
. The invention accordingly comprises the several steps and the relation of one or more of such steps with respect to ~ ;
:` each of the others thereof~ which will be exemplified in the method hereinafter disclosed, and the scope of the invention will b~ indica~ed in the claim~.

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D~TAILED D~SCRIPTION OF THE INVENTION
Enzymatic preparations that may be employed in the invention are easily obtained by destroying the cell wall of ~
the microorganism and extracting the water-soluble fraction ~ ~;
therefrom. Numerous methods and extraction techniques are known to the art-skilled and any oneof these conventional methods may be employed. Preferred extraction techniques, however, within the scope of this invention are those in which no additional chemical contaminants are introduced into the cells, iOe. primarily mechanical opening methods, After destruction of the microorganism cell wall, the ;~
soluble raction can be used as such within the scope of the method according to the invention. It is desirable to separate the insoluble fraction thererom, but it is not absolutely necessary to do so. Pre~erably the enzymatic preparation is obtained by adding conventional precipitan~s to the soluble fxaction after separation of the insoluble fraction therefrom.
Because of its availability, ammonium sulfate is a preferred precipitant, and precipitation of dissolved enzymatic protein of up to 70% ammonium sulfate saturation was found to be particularly effective.
Surprisingly, commercial enzymatic preparations which are employed as detergent additives may be used according to the method of this invention. Such enzymatic preparations are ; manufactured on a large scale and are therefore comparatively lnexpensive, "( 1, .

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The quantity of enzyme which must be added to the effluent containing water depends upon the condition of the water to be processed and on the enzymatic preparation employed therein.
Specifically, the type and amount of contamination~ the source -of the enzymatic preparation and the method used in its manufacture are taken into account to determine the quantity of ¦ enzyme employed. The quantity of enzyme employed also depends ¦ upon whether decomposition is to be effected by the enzymatic preparation alone9 or whether the enzymatic preparation is being employed in conjunction with the live microorganisms ound in the conventional biological decomposition methods. ~-In the latter case it is, for instance, possible to use the instant method when the process facility is inadequate for instance when an unusually large vol-ume of waste water is to be processed or when-there is a sudden increase of contamination `;
in the water under treatment which exceeds the usual process or contamination rate. By supplementing the action of a conventional ~acility according to this invention, it is -possible to substantially increase the decomposition of the contaminants and thus obtain the benefit of a capacity increase ;1 without actually increasing the capacity of the facility.
In general it is necessary to add between about .1 mg and about ,5 mg of enzyme per liter of water under treatment. ~;
However, the amount of enzyme which is added may vary according ;
to the makeup of the water under process. ~ ~
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In the application of the method of the invention, it has been observed that the addition of enzymes within the scope of the invention to water containing other microorganisms ~- -which ordinarily decompose contaminants increases the decomposition rate kontaminants in the water and promotes the growth of the microorganisms therein. According to one particular embodiment of the invention, this effect is ;-enhanced by further adding to the water under treatment nutrients for the microorganisms present therein. Suitable nutrients include carbon sources, such as carbohydrates, amino acids, mineral salts and ~he like. While these additives increase the quantity of organic contamination in the water, ; they eoncomitantly increase the activity of the microorganisms which decompose the effluent The method of the invèntion substantially accelerates the decomposition of the contaminants in the water, which is reflected in a corresponding increase in oxygen consumption. ~ ~
When the method o the invention is used, therefore, it is -preferable to aerate the water in process. The method of the invention is therefore particularly suited to combination with methods in which contaminated water is aerated, for instance the trickling filter method, the submerged trickling filter ;~
method, and the activated sludge method with àeration by compressed air, surface aeration and combined aeration methods.
According to the invention it is possible to accelerate , l . I

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decomposition of organic effluents in wa~er processed through conventional purification facilities of a given size and increase the processin~ capacity of existing facilities without structurally changing existing facilities and without improving the quality of the water in process. The increased decomposition activity obtained according to the invention provides a reduction of the quantity of sludge generated by the process. If no microorganisms are used to decontaminate the water, then the generation of sludge may even be completely eliminated.
The examples that follow are for illustrative purposes only,and ~
are not intended to limit the scope of the invention. ~ ~ ;

A. An enzyme was extracted as follows. ~ 400 ml sample ~ ;
of lake water from a badly contaminated lake (Edeberg Lake near Plon~ was taken and employed as a source of microorganisms for inoculating 10 liters of a commercial nutrient solution ~ ;
(ordinarily for microorganisms of a meat extract) and a broth (Difo Nutrient Broth).
, 20 The inoculate was incubated for 6 days at 27C. The ;;
bacterial culture obtained thererom was removed by centrifuging ~ ;
and washed twice with Tris buffer pH 7.8. The washed cells were then broken up at low temperature with ultrasonic waves.
The nonsoluble cell wall fragments were removed by centrifuging and the clear remnant was brought to 60% saturation with ,.,,, ' ' _g_ .-:
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1041J~81 ammonium sulfate solution. The precipitate was removed by centrifuging, and ammonium sulfate was added to the clear '~
remnant until 70% saturation was obtained. The precipitates obtained in this manner, respectively referred to as 60%
precipitate and 70% precipitate below, were absorbed with Tris buffer pH 7.8 and used for the subsequent experiments.
B. Synthetic water, consisting of water from the Edeberg Lake having added thereto .15 mg glucose per liter and .15 mg glutamic acid per liter was used for a series of tests. The decomposition of the contaminants, with and without additions ~: ~
of the enzymatic preparation was observed by reference to ~;
oxygen consumption in the samples.
The tests were conducted in 100 ml Erlenmeyer flasks.
, Oxygen was measured after 0, 2 and 4 days, The temperature ; was 27C, Experimental procedure and results are shown in table 1.
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,~ ~ TABLE 1 O Days Test No. mg 02/1 Average . 20 -1 lloO ~ 1 ~ 2 .

,', 3 11.2 3 11.3 4 11.4 10.6 10.4 6 10.3 7 10.5 ~-8 10,6 ,,. ' -10- , ,j , ,.. . .. .. . .

2 Days Test No. mg 02/1 Average02-consumption ~ :
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1 6,4 6.3 4.8 ;~
2 . 6.3

3 6.7 6.7 4.6 '~ :
0.0 000 10.5 6 0.0 7 0.3 ~:~
8 0.3 0 3 10 03

4 Days .
Test No. mg 02/1 ~verage02-consumption _ _ ~. ...........
1 204 203 8.8 2 2.3- '~
3 200 2.0 9.3 ~:~
- 0.0 . 0,0 10.5 : 6 000 : 7 0.0 :~
8 0 0 10.6 Notes `~ .
. Tests 1 and 2: Water from Edeberg Lake Tests 3 and 4: Water from Edeberg Lake + .15 mgil glucose + .
. .15 mg/l glutamic acid Tests 5 and 6: Same as tests 3 and 4 + .1 ml 60% enzyme ;:;
preparation ..
Tests 7 and 8: Same as tests 3 and 4 + .1 ml 70% enzyme preparation.
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~L0416131 The above values show that according to the invention all oxygen has already been consumed after 2 days. In the control ~¦ -tests without enzyme (tests l to 3), oxygen is present even after 4 days.

The tests o example 1 were repeated, but the additive was an enzymatic preparation which had been immedia~ely precipitated to a saturation of 70% ammonium sulfate. These three tests were made with: Water from Edeberg Lake (test 8)~ water from Edeberg Lake + .15 mg glucose ~ .15 mg glul-amic acid per liter (snythetic waste water; tes~ lO) and snythetic waste water according to test 8 ~ .1 ml en~yme preparation. The number of microbes was observed for each test series at the start and during the next 4 daysO The results are shown in table 2 below:
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Test No. Number of microbes per ml. 103 11 2268 3534 4063 111~ 1771 _ _ _ __ O From the above figures for microbes the following values , for the duration of a generation can be computed:
.~!Test 9 Hours 186 .. , l !

~ 4 1~ 8 The above tests show that the number of microbes is clearly increased by ~he method in accordance with the invention in comparison with the control tests and that the duration of a generation has been correspondingly reduced. The protein content of the bacterial biomass which is obtained by the~ ;~
method according to the invention is clearly higher than the quantity of protein which had been added in the form o the ~ ~
enzymatic preparation. This can only be explained by increased ~ ;
decomposition. -,.'.,: ,';
EXAMPLE 3 -~
The decomposition velocity of organic substances in synthetic waste water was investigated as a function of th addition of enzymes thereto. Decomposition was performed in a ~ -laboratory scale model of a purification facility. This model is described in the periodical "Das Wasser" vol. 40, pages 369-389, 1973. It consists of an areation chamber and a sedimentation basin and thus corresponds to a conventional ¦activated sludge plant. Capacity is 3 liters.
The dissolved organic materials were determined by dichromate ;
oxidation after filtering through a diaphragm filter ( .45/u).
(cf. for instance, J.D.H. Strickland and T.R. Parsons "A
Practical Handbook of Seawater Analysis", Fisheries Research Board of Canada, Bulletin 167, Ottawa 1972, pp. 207-211). This pro~edure gives the quantity of dissolved organic compounds in mg carbon per liter.

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In one test the enzymes were bacterial enzymes obtained from waste water; in the other test a commercial enzyme ¦
preparation manufactured by the Merck Company3 Darmstadt, was used. The commercial enzyme preparation is the type of preparation which is, for instance, added to detergents with enzyme action. Preferred are preparations which are particularly suited for the decomposition of protein and blood. Such preparations are usually designed for use in industrial rinsing machines. The preparation used in this instance is "7570 Extran ~P 41 enzymatic". ;
The results of the tests are summarized in table 3.
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. . _ Decomposition of snythetic waste water without and with addition o bacteria and enzymes Time g C/l Decompo- mg C/l % mg C/l % ¦ mg C/l %
i sition in h ' in % , ' ~ :~
~0 65.5 0 61.0 0 1 ~5.90 1 58.6 0 17 , 6000 8,~ ; 61.0 0 64.12.8 58.2 007 ' 1 6000 804 59.1 3.2 58.611.81 5707 1.5 241 60.0 8.4 58.2 4.6 54.5 17.3 55.5 5.3 ,' /~81 61.4 6.3 1 56.4 7.6 27.7 58.6 27,3 53.4 72' 62.4 4.7 ~ 55.4 9.2 18.2 72.4 19.5 64.8 96 61.4 6.3 1 55.7 8.7 15.9 75.9 11,8 79.9 :' ....
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¦ In this table: 1041681 ~ ~

Column 1 = synthetic waste water (lake water with .lS mg glucose ¦ ~ .15 mg glutamic acid per liter added) ~Column 2 = same as 1, with addition of isolated bacteria taken from a body of water (strain 8 of our collection);
quanti~y added a 3 x 105 cells/ml; Strain 8 isArthobact er sp. 20124 and a deposit thereof has been made in the , public depository of the German Collection of Micro- -organisms, an ar~-recognized depository at D-34 ~ -I lO ~ Goettingen Gresebackstr. 8.
Column 3 = same as 2, with 30 mg nitrogen enzyme added per liter (60% ammonium sulfate precipitate as above);
¦Column 4 = same as 2, with 30 mg Extran (Merck) per liter added.
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The table shows that in synthetic waste water (column 1) and ~ ?~

in synthetic waste water + bacteria (column 2) there is practical ¦ `~

ly no decomposition. After enzymes are added (columns 3 and 4), ~-, h~wever, very intensive decomposition occurs. After four days, ¦ 75.9 and 79.9% respectively of the dissolved organic material are ¦ removed from the waste water.
! EXAMPLE 4 ~ ¦ The tests were conducted as in example 3, but 10 mg Extran ¦ ¦ (Merck)(column 3) and 30 mg Extran (Merck) (column 4) were added I to the yntbetic waste water.

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Decomposition of synthetic waste water without and with addition of bacteria and enzymes ~

Time mg C/l Decompo- mg C/l % mg C/l %mg C/l % ~; .
sition :
in h in % ~ :
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:
0 92.9 0 7805 0 97.6 0 7806 0 ~ `
2~ ~2~7 0,2 95.2 +12.1 7203 26 60.9 22.6 lo 4~ ~8.6 4.7 8507 +10.9 31.0 68.3 2104 72.8 72 85.7 7.8 81.9 +10.4 7.1 92~8 16.7 79.8 96 85,7 7.8 8100 +10.3 5.2 94.7 11.9 8~.9 120 85.2 8.3 80.5 ~10.3 5.2 94.7 11.9 84.9 144 8500 806 78.6 + 1.0 502 94.7 lQ.5 86.7 :
168 85.0 8.6 73.8. 0 5.2 94O7 10.5 86.7 -.
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: ~ : Column L = synthetic waste water (lake water with .15 mg glucose ; :
and + .15 mg glutamLc acid per liter added); :

. . Column 2 = same as 1, ~ith isolated bacteria from bodies of ~:~
.~ 20 water added (strain 8 o~ our collection); quantity -:
added = 1 x 10 cells/ml, Zolumn 3 - same as 2 + 10 mg Extran (Merck) per liter;

Column 4 = same as 2 + 30 mg Extran (Merck) per literO .
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Table 4 shows that without the addition of enzyme decomposition starts very slowly. If bacteria are added ~ ~
(column 2), however, they increase the quantity of ~-the dissolved organic material (extra-cellular metabolism ~ I
products). By the addition of enzymes almost complete decomposition is obtained. The decomposition is characterized .
by a substantial reduction of the quantity of dissolved organic material.
It will thus be seen that the objects set forth above, among ~hose made apparent from the preceding descriptionj are efficiently attained and, since certain changes may be made in carrying out the above process without departing ~rom the ;~
spirit and scope of the invention, it is intended that all I matter contained in the above description sha~ll be interpreted aB illustrative and not in a limiting sense.
~1~ It is also to be understood that the following claims are intended to cover all of the generic and speciic features - o the invention herein desc~ibed, and all sta~ements of the scope of the invention which, as a matter of language, might ¦ be said to fall therebetween. ~ -~
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Claims (10)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A method for purifying an effluent containing water comprising introducing an enzyme of a microorganism having an aqueous ambience into said effluent containing water.

2. The method as claimed in claim 1 wherein the enzyme is characterized as a detergent enzyme.

3. The method as claimed in claim 1 including extracting the enzyme from a microorganism whose ambience is the effluent containing water under treatment.

4. The method as claimed in claim 1 including obtaining said enzyme by fracturing the cell wall of said micro-organism, separating the soluble fraction thereof from insoluble fragments, and adding an enzyme precipitant to said soluble fraction.

5, The method as claimed in claim 4 wherein the enzyme precipitant is ammonium sulfate and including precipi-tating soluble enzymatic protein from said soluble fraction of up to 70% saturation with ammonium sulfate.

6. The method as claimed in claim 1 including introducing nutrients into the effluent containing water under treatment.

7. The method as claimed in claim 6 wherein said nutrients include carbohydrates and amino acids.

8. The method as claimed in claim 1 including oxygenating the effluent containing water under treatment.

9. The method as claimed in claim 1 including introducing between about .1 mg and about .5 mg of said enzyme per liter of effluent containing water under treatment.

10. The method as claimed in claim 4 including prior to fracturing the cell wall of said microorganism, inoculat-ing a nutrient medium with said microorganism, incubating the inoculate at about 27°C, centrifuging the culture, and washing the culture in a buffered solution.