CA2046699A1

CA2046699A1 - Continuous bacterial mutation process

Info

Publication number: CA2046699A1
Application number: CA 2046699
Authority: CA
Inventors: Gensen Kai; A. Scott Weber; Wei-Chi Ying
Original assignee: Individual
Current assignee: Occidental Chemical Corp; State University of New York SUNY
Priority date: 1990-07-26
Filing date: 1991-07-10
Publication date: 1992-01-27
Also published as: GB2247012A; DE4124900A1; GB2247012B; FR2665180A1; GB9115086D0; JPH0568997A; FR2665180B1

Abstract

ABSTRACT OF THE DISCLOSURE

CONTINUOUS BACTERIAL MUTATION PROCESS
Disclosed is a method of continuously biologically acting on a target chemical in an aqueous stream by cultivating bacteria under non-growth-limiting conditions, exposing the cultivated bacteria to mutation-producing ultraviolet light, and exposing the mutated bacteria to the target chemical under growth-limiting conditions. Also disclosed is apparatus for performing this method.

Description

20~6~9 Case 6205 RDF/kmf CONTINI~OUS BACTERIAI. ~IUTATION PROCESS
Background of the. Invention This invention relates to a method of continuously biologically acting on a target chemical in an aqueous stream.
Specifically, it relates a process in which bacteria grown under non-growth-limiting conditions are exposed to mutating ultra-violet light and then to a target chemical under growth-limiting conditions.
The presence of toxic chemicals in the environment has become a matter of increasing concern and is now the subject of numerous laws and regulations. The removal or destruction of these chemicals can constitute an enormous cost, especially when the chemicals are dispersed and are present in low concentrations. One method of remediating such chemicals is by means of microorganisms that seek out and biodegrade them.
It is known that bacteria that live in the presence of recalcitrant or persistent chemicals, such as some halogenated organic compounds, can develop a tolerance for the chemicals and may even be able to biodegrade them. Genes that encode enzymes which degrade the chemicals formed at each step in a degrative pathway can be assembled by genetic engineering and inserted into a single organism. The mutation of bacteria has also been found to be useful in producing bacteria of enhanced capability for biodegrading such chemicals. Once a useful 06/15/gO

204~fi99 bacteria has been isolated and cultivated, it can be added to a site contaminated with the chemica:L in order to biodegrade it to less toxic or non-toxic chemica:Ls.
However, there are a number of situations where these techniques are not useful. For example, in an industrial waste stream, a mixture of target chemicals may be present, and their composition and concentrations may change from time to time as the industrial processes change. In such a situation, a bacteria tailored to attack a specific target chemical may be entirely useless against other target chemicals. Also, where the waste stream is moving, the bacteria may be flushed out at a rate faster than their rate of growth in the waste stream and, therefore, they may disappear from the waste stream in a short time. Some target chemicals must be degraded in several steps and a different bacteria may be required for each step.
Under these circumstances, if the mixture of bacteria are injected as a batch, the bacteria that perform the later degradation steps may die or be flushed out before the bacteria that perform the earlier degradation steps have produced the chemicals that are used in the later steps. For these and other situations, it would be desirable to have a process for continuously producing mutated bacteria that are capable of acting on the target chemicals that are actually present.
Summary of the Invention We have discovered a method and apparatus for continuously biologically acting on target chemicals in an aqueous stream.
In the method and apparatus of this invention, mutated bacteria are continuously being produced for the purpose of acting on 20~66'~9 the target chemical. Unlike a batch process, the continuous process of this invention can be operated to optimize the essential steps of growth of the feed population, W-induced mutation, and stabilization and mu:Ltiplication of the mutant bacteria. This invention overcomes many of the difficulties encountered when bacteria are added as a batch to the target chemical. For example, because the mutated bacteria are continuously being produced, should the target chemical change, newly mutated bacteria which can survive and grow on this new compound will be selected. The use of continuously produced mutated bacteria permits the injection of the bacteria even into streams from which they are flushed out at a higher rate than they can grow. And, if the target chemical degrades in steps, mutated bacteria will be selected for which can attack the chemicals produced in the later steps as they are produced.
The continuous process of this invention enables one to produce a large number of mutants and to automatically screen for the best strain at a much lower volumetric requirement.
For example, if the residence time of a bacteria in the continuous W mutation chamber of this invention is 1/25 of a day, then it would take a tank 25 times larger to produce the same number of mutated bacteria in a batch process. Finally, if the degradation of the target chemical requires a radical change in the bacteria, it is unlikely to occur in a batch system because in a batch system a radical change must occur all at once. But, in the continuous process of this invention, instead of a single radical change occurring in the bacteria, a series of small steps can occur, which is much more probable.

2 ~
As a result, radically changed bact:eria are more easilyproduced in the process of this invention.
Description of the Invention Figure l is a diagrammatic view of a certain presently preferred embodiment illustrating the method and apparatus of this invention.
Figure 2 is a graph giving the relationship between irradiation time and mutation frequency (see Example 1 which follows).
Figures 3 and 4 are graphs giving the relationship between time and p-CBA concentration tsee Example 1 which follows).
In FigurP 1, a source of bacteria is provided through line ' to reactor 2. Examples of sources for the bacteria used in this invention include municipal wastewater, activated sludge, soil inoculation from contaminated sites, or the purchase of suitable bacteria. It is preferable that a variety of bacterial species be supplied to reactor 2 in order to maximize the likelihood of forming useful mutations. It also is preferable to use bacteria that are indigenous to an environment in which the target chemical is present as these bacteria may be genetically closer to the desired mutated bacteria than bacteria that live in a different environment.
Reactor 2 may constitute a sludge treatment facility, or it may be a special reactor created for the purpose of this invention.
A source of organic carbon for the bacteria and a source of nitrogen and phosphorus for the bacteria flow from feed tanks 3 and 4 through lines 5 and 6, respectively, to first reactor 2. The nutrients provided through tanks 3 and 4 are 204~9 conventional and it is simply necessary that sufficient nutrients be supplied to reactor 2 to enable bacteria in reactor 2 to grow under non-growth-limiting conditions (i.e., the time that the bacteria are present in the reactor is insufficient for the supply of nutrients that are present to limit their growth). Non-growth limiting conditions are needed so that the bacteria are replicating when they leave reactor 2 as replicating bacteria are more vulnerable to mutation during replication.
Also, if insufficient nutrients are provided to reactor 2, the bacteria will tend to agglomerate, which will also make them less susceptible to mutation. Of course, the temperature and pH should be suitable for growth of bacteria, as is well-known in the art.
From reactor 2, the replicating bacteria pass through line 7 to mutation chamber 8 where the bacteria are exposed to ultraviolet (W) light, which can be, for example, at a wave length of about l9O to about 300 nm. The time of exposure to the W light should be sufficiently long to induce mutations but not so long as to kill the bacteria. Actual time of exposure will depend on the W light source used. From mutation chamber 8 the bacteria pass through line 9 to second reactor lO.
Reactor 10 greatly adds to the efficiency and effectiveness of the process of this invention and is especially needed to initiate the degradation process. After degradation has begun, reactor lO is generally less necessary to obtain the full benefits of this invention. The purpose o~

2046~

reactor lO is to stabilize the population of bacteria by enabling them to repair whatever damage was done in mutation chamber 8 and to replicate, preferably at least two generations; preferably, the bacteria remain in reactor lO for at least two h~urs to permit the population to stabilize and to replicate. It is preferable that sufficient nutrients be provided to reactor 2 so that not all the nutrients are used up in reactor 2, but some pass through mutation chamber 8, enabling the bacteria in reactor lO to grow under non-growth-limiting conditions. (~dditional nutrients can, of course, be added directly to reactor lO if needed.~ The bacteria leave reactor lO through line 11 and enter the third reactor 12.
A target chemical from tank 13 is supplied through line 14 to reactor 12. While it is preferable to contact the bacteria with the target chemical only after they have mutated, it is also possible to contact the bacteria with the target chemical before mutation by, for example, adding the target chemical to reactor 2. For the maximum production of mutations, it is desirable that the concentration of the target chemical in reactor 12 be at a non-inhibitory level, however, so that high bacterial growth rates can occur.
The target chemical typically will be a halogenated organic, for example, a dioxin or PCB (polychlorinated biphenyl). The mutated bacteria may be able to use it as a source of carbon and ener~y while the unmutated bacterial will not. However, the target chemical may also be a chemical that one wishes to convert and/or to extract using the bacteria.

2~6~
Bioextraction may be accomplished with various metals, such as gold or radioactive strontium, and non-metals, such as sulfur or other inorganic substances, for the purpose of either removing them from the environment or recovering them.
Bioextraction may involve solubilizing the target chemical, insolubilizing the target chemical, volatilizing the target chemical, incorporating the target chemical into the bacteria, or another process.
In reactor 12 the bacteria are grown under growth-limiting conditions (i.e., the time that the bacteria are in the reactor is long enough that their growth becomes limited by the supply of nutrients). Insufficient nutrients are present for all the bacteria to grow at their maximum rate, so that those bacteria that are capable of growing in the presence of the target chemical, or preferably, utilizing the target chemical, survive and replicate while the populations of competing bacteria are reduced. From reactor 12 the successful mutated bacteria can be continuously removed and sent to a treatment facility through line 15 or can be otherwise utilized. Reactor 12 could also itself constitute a waste-treatment plant already containing the target chemical. Thus, bacteria could be removed from waste-treatment plant 12 to reactor 2, sent through the mutation chamber 8 and returned after reactor lO to the waste-treatment facility (reactor 12) once again as a continuous process.
Figure 1 also shows additional apparatus that was utilized in the examples hereinafter described to establish the effectiveness of the process of this invention. In order to 204~6~5 compare mutated bacteria with unmutated bacteria, unmutated bacteria can be passed through line 16 to reactor 17 after dilution with water from tank 18 (to maintain equal population densities in reactors 12 and 17) which passes through line 19.
The target chemical can be supplied to tank 17 from line 20 and bacteria can leave reactor 17 through line 21.
The following examples further illustrate this invention.

The purpose of this experiment was to determine whether W
light would mutate a continuously flowing population of bacteria.
The experimental apparatus consisted of a plexiglass reactor (2 in Figure 1) and a W mutation chamber ~8 in Figure 1) containing one of two models of UV lamp from Spectronics Corporation, Model llSC-l with an average lamp intensity of 4,500uw/cm2 and Model llSC-2 with 2,000uw/cm2 at 254 nm, at 1.91 cm. The detention time in the mutation tube determined the irradiation dosage.
The composition of feed solution for the cultivation reactor was a basic medium supplemented with 1.25 g/L fructose:

2 ~ 9 Composition of the Mineral Nutrient Stock Solution CONSTITUENT AMOUNT
Conc. H2SO4 10mL
2(So4)2 nH2 8.6g MnC12.4H2O 2g ZnSO4 0.81g CuSO~.5H2O 0.81g CoC12.6H2O 0.24g Na2MoO4 0.2lg H3BO4 0.08g Distilled Water to 1 L
Composition of Basic Medium CONSTITUENT AMOUNT
(NH4)2SO4 0.2g MgSO4.7H2O 0.2g C 4 2 0.01g M.N.S.S.* 0.5mL
KH2P04/Na2HP04 Buffer (pH7.2) 0.04M
EDTA
(disodium salt) 5 x 10 6M
Distilled Water to lL
*M.N.S.S. Mineral Nutrient Stock Solution To quantify the continuous flow mutation device, the mutation frequency of the system was determined. Mutation frequency, the number of mutants divided by the number of survivors, was determined as a function of radiation dose.
Mutants were identified by their ability to grow on plates _ g _ 20~9~

containing the antibiotic streptomycin. The mutation frequency as a function of irradiation time was compared to the natural mutation frequency of the population used for these experiments. The natural mutation frequency was found to be 1.14 x lO 7. ~s shown in Figure 2, an optimal mutation frequency for the model llSC-1 lamp was determined to occur at a time of approximately 55 seconds, and this value corresponded to 3.5 x lO 4, significantly higher than the natural mutation frequency. A comparable increased mutation frequency was found for the other lamp investigated with an optimal value at approximately 25 seconds~
Exam~le 2 In this experiment the continuous flow mutation apparatus consisted of the apparatus used in Example 1 followed by reactors as shown in Figure 1. The plexiglass reactors were arranged in series with the W mutation chamber located between the first reactor Rl (2 in Figure l~ and the second reactor R2 (lO in Figure l).
The fourth reactor R4 (17 in Figure 1) was fed a comparable amount of bacteria as R3 (12 in Figure 1) using R1 as the inoculating source. Because the concentration of bacteria in Rl was greater than the R2, the feed from Rl to R4 was diluted with distilled water. Detention times in R3 and R4 were maintained the same by controlling feed rates to these reactors. The target compound concentration in R4 was equivalent to R3.
All reactors were well mixed and aerated with humidified air. Delivery of flows between the reactors was accomplished 204~
with the use of positive displacement pumps ~hrough vinyl tubingO Other parameters pertinant to the volume, feed rate, and detention time and some conditions are summarized in the following table:
REACTORSVOLUME FLOW ~T~DETENTION TIME
_ mL __ mL/min _hours_ _ R 1 1420 9 2.63 R 2 1230 5.17 3.97 R 3 9550 5.74 27.73 R 4 9550 5.74 27.73 W Chamber 5 5.17 0.016 When a relatively stable state in Rl had developed, the cultivated population was characterized by multiple strains, exponential phase growth, a viable cell count of approximately 108 to 109 colony forming units (CFUs)/mL of culture fluid, and an aqueous phase total organic carbon (TOC3 concentration of about 200 mg/L. A viable cell count of 108 to 109 CFU/mL is favored to ensure adequate amounts of bacteria for the mutation experiment. The residual aqueous phase TOC concentration is needed for mutant growth in R2.
The experimental system was run to test the quantitative requirement of mutants for obtaining a p-chlorobenzoic acid (p-CBA) degrading strain. Mixed culture bacteria from Rl was stirred and irradiated in the W chamber then pumped into R2 to screen for the desired mutant. At O hour, R3 and R4 were filled up with the basic medium and 100 mg/L of p-CBA. The p-CBA stock solution of 1 g/L was continuously provided with the culture amount from R2, and the concentration of p-CBA was ~O~g~99 measured periodically in R3, where selection proceeded, and in R4, which functioned as the contro:L for the continusus mutation scheme.
The following table shows that p-CBA degrading mutants were evolved in the mutation system:

Time p-CBA Chloride hours mg/L mg/L
Sample Control Sam~le Control o 99.8100.2 0.6 0.6 12 100.2107.2 3.4 1.9 24 98.6105.8 4.5 2.3 36 93.~ 98.5 5.2 2.2 48 83.9106.1 7.4 2.4 ~0.6105.6 7.6 2.4 72 82.4112.2 8.6 2.3 96 39.6103.4 12.2 2.4 110 4.2 98.0 21.7 2.5 144 3.8 99.0 22.5 2.8 It seemed that at least two types of mutant were created during the course of the experiment. The mutant that appeared in the first 70 hours had a relatively low biodegradation speed while the second type of mutant, which appeared afterward, had a much faster speed.
Using the same bacterial source, a batch study in flasks was conducted. The total batch samples were divided into two sets. Set A was inoculated with 5 mL of mutant culture taken from R2, while 50 mL mutant from the same source was inoculated into Set B. Seven replicates were made for each set. Also, a 2~46~"9 comparable amount of non-irradiated bacteria from Rl were used as control for the batch study. It was expected that biodegradation would be observed in some of the flasks so a statistic calculation could be made to estimate the probability of mutant requirement in this experimental condition. However, no biodegradation was observed in any of the 385 mL mutant cultures.
To justify the function of the R2 reactor, the system was run without R2. As shown in the Figure 3, no significant biodegradation of the p-CBA was observed during the time when R2 was absent. Figure 4 shows that the R2 reactor was very helpful in initiating biodegradation of the p-CBA, once the biodegradation was fully established, the R2 reactor could be withdrawn from the treatment train without adversely affecting treatment performance.
Although it is possible that the desired mutant might appear sometime after 72 hours, it can be concluded that the system is at least more efficient with R2 than without.
In additional experiments, the system was tested for evolving populations capable of degrading p-CBA, 2,4-dichlorobenzoic acid (2,4-DCBA), and chlorendic (HET) acid [1,4,5,6,7,7-hexachlorobicyclo[2,2,1]-hept-5-ene-2, 3-dicarboxylic acid; CAS Number 115-28-6]. These experiments differed from those described earlier in that populations tested were obtained from R3. Control populations were obtained from R4. Neither R3 nor R4 received the target compound. Populations obtained from R3 and R4 of the system were mixed with basic medium and target chemicals, 204~69~
respectively, in 500 mL flasks and placed in mixed shakerflasks.
All mutant samples showed biodegradation, which was confirmed with COD (chemical oxygen demand) and chloride tests.
P-CBA biodegradation was completed in less than 25 days for each of the mutants replicated, and 2,4-DCBA in around 35 days.
There was some inconsistency in the P-CBA control data obtained. For one control flask, the control population initiated p-CBA biodegradation at about 25 days. A slower rate of biodegradation was observed for this control relative to the rate observed for the mutated populations. Thus, the degradation that occurred in this control sample can be considered to be a spontaneous event. No biodegradation of p-CBA wa~ observed in the other control during the 65 day test period.
Inspection of chlorendic acid biodegradation results revealed that the mutated cultures brought about a reaction not achieved by the non-mutated controls. Evidence of this reaction was in the form of increased relative absorbance measurements. In three of the mutated replicates the reaction was initiated after approximately 4 days of reaction time.
Increa~ed relative absorbance was observed for the fourth mutated replicate on day 8. No increase in relative absorbance was noted for either of the controls.

To further assess the likelihood of chlorendic acid biodegradation resulting from the mutated populations, the concentrations of chlorendic acid, TOC, and chloride in two replicates were measured after 20 da~s of reaction time. The 2 ~

results from these analyses are reported in the following table.

Chlorendic Acid TOC Chloride mq/L m~3/Lmg/L
Time (day) 0 20 o 20_0_ _ 20 H 1 210 166 58 450.35 7~22 H 2 210 145 58 430.35 5.64 During the 20 days test period, chlorendic acid concentration dropped from 210 mg/L to 166 and 145 mg/L for populations H 1 and H 2, respectively, which represents a 21 and 31 percent decrease in chlorendic acid. Total organic carbon concentration also dropped during the same period from an initial concentration of 58 mg/L to 45 and 43 mg/L for H 1 and H 2, respectively, which represents removal percentages of approximately 22 and 26 percent. Additional evidence of chlorendic acid biodegradation can be seen from the chloride release data. Chloride was released in all test flasks containing mutant cultures and averaged 7.34 mg/L Cl .

Claims

1. A method of continuously biologically acting on a target chemical in an aqueous stream comprising (1) cultivating bacteria under non-growth-limiting conditions;
(2) exposing said cultivated bacteria to mutation-producing ultraviolet light; and (3) exposing said mutated bacteria to said target chemical under growth-limiting conditions.

2. A method according to Claim 1 wherein said target chemical is present in step (1).

3. A method according to Claim 1 wherein said target chemical is not present in step (1) or step (2).

4. A method according to Claim 1 wherein said target chemical is a halogenated organic compound.

5. A method according to Claim 1 including, between steps (2) and (3), the additional step of replicating said mutated bacteria at least two divisions under non-growth limiting conditions.

6. A method according to Claim 5 wherein said replication occurs for at least two hours.

7. A method according to Claim 1 wherein said bacteria are obtained from a source containing said target chemical.

8. A method according to Claim 1 wherein the concentration of said target chemical in step (3) is insufficient to inhibit the growth of said bacteria.

9. A method according to Claim 1 wherein said bacteria in step (1) are obtained from a biological reactor containing activated sludge, said target chemical is one or more biodegradable compounds in said activated sludge, and said mutated bacteria from step (3) are returned to said biological reactor.

10. In a process where sludge is digested in a biological reactor by an indigenous population of bacteria, an improved method of enhancing the biodegradation of halogenated organics in said sludge comprising (1) continuously passing a portion of said sludge containing said bacteria from said biological reactor to a first reactor where the growth of said bacteria is stimulated;
(2) passing said sludge from said first reactor to a W
mutation reactor where the bacteria in said sludge are exposed to W light, causing mutation of said bacteria;
(3) passing said sludge from said UV mutation reactor to a second reactor where the growth of mutated bacteria is stimulated; and (4) returning said sludge from said second reactor to said biological reactor.

11. Apparatus for continuously biologically acting on a target chemical in an aqueous stream comprising (1) a first reactor for cultivating bacteria under non-growth-limiting conditions (2) means for exposing said cultivated bacteria to mutation-producing ultraviolet light; and (3) a second reactor for exposing said mutated bacteria to said target chemical under growth-limiting conditions.

12. Apparatus according to Claim 11 including means for replicating said mutated bacteria at least two divisions prior to exposing said mutated bacteria to said target chemical.

13. Apparatus according to Claim 11 including (1) a biological reactor for the digestion of activated sludge by said bacteria;
(2) means for diverting a portion of said activated sludge from said biological reactor to said first reactor; and (3) means for returning said mutated bacteria to said biological reactor.