WO1993022247A1

WO1993022247A1 - Biological degradation of methylene chloride

Info

Publication number: WO1993022247A1
Application number: PCT/US1993/003876
Authority: WO
Inventors: Domenic A. Paone; Patrick J. Evans; Marika A. Suominen-Yeh
Original assignee: Celgene Corporation
Priority date: 1992-04-24
Filing date: 1993-04-23
Publication date: 1993-11-11

Abstract

Processes, apparatus, and materials for the microbiological degradation of methylene chloride to carbon dioxide and hydrogen chloride are described. The process employs a biodegradation matrix in which a methanol-grown methylotrophic bacterium is affixed by oxygen-promoted secondary cell attachment to particulate activated coal-derived charcoal.

Description

BIOLOGICAL DEGRADATION OF METHYLENE CHLORIDE

The present invention relates to processes and materi¬ als for use in the biological degradation of methylene 5 chloride.

BACKGROUND OF THE INVENTION

Methylene chloride, an industrial solvent widely used in the pharmaceutical, plastics, film, and other industries, is considered a suspect carcinogen on the EPA Priority 10 Pollutant List.

In order to reduce emissions, methylene chloride as a liquid often is adsorbed onto granular activated carbon which then is incinerated. This is not particularly effi¬ cient since at concentrations of 500 to 5000 ppm, typically 15 loadings of only 20 to 75 mg/g of activated carbon are obtained. Vacuum extraction also has been employed in some instances where ground water is contaminated with methylene chloride.

The ability of certain microorganisms to degrade 20 methylene chloride in a laboratory environment has been well documented during the past decade.

Rittmann et al . , Appl. Environ. Microbiol., 39: 1225- 1226 (1980) , reported suspended and fixed-film bacteria from primary sewage effluent could utilize methylene chloride 25 (dichloromethane) as the only exogenous energy source.

^•

Brunner et al . _r Appl. Environ. Microbiol., 40: 950-958 (1980) , described the bacterial degradation of methylene chloride with a facultative methylotrophic bacterium iso- lated as an airborne contaminant and believed to belong to the genus Pseudomonas.

Stuc i et al. _t Arch. Microbiol., 130:366-371 (1981), reported on the isolation and characterization of a strain of Hyphomicrobi m, the cell extracts of which exhibited glu- tathione-dependent conversion of methylene chloride to formaldehyde and hydrochloric acid.

Klecka, Appl. Environ. Microbiol., 44: 701-707 (1982), examined the fate of methylene chloride in activated sewage sludge, postulating that initial attack in the observed biodegradation might involve either a monooxygenase or hal- idohydrolase.

Kohler-Stab et al . , J. Bacteriol., 162:2, 676-681 (1985) , purified a dehalogenase obtained from Hyphomicrobium sp. strain DM2 which Stucki et al . , supra, had shown effected glutathione-dependent metabolism of methylene chlo¬ ride.

LaPat-Polsko et al . , Appl. Environ. Microbiol., 47: 825-830 (1984) , reported a strain of Pseudomonas sp. strain LP, was capable of simultaneously consuming two substrates, methylene chloride and acetate, at different concentrations. The authors employed a biofilm on glass beads and observed that removal of methylene chloride was consistently higher in the presence of acetate.

Kohler-Stab et al . , J. Gen. Microbiol., 132, 2837-2843 (1986) , examined methylene chloride dehalogenases obtained from two Hyphomicrobium strains and two Pseudomonas strains and concluded they shared homologous N-terminal a ino acid sequences (sequenced up to residue 15) and operated by a uniform dehalogenase enzyme pattern. Galli et al . , J. Gen. Microbiol., 134, 943-952 (1988), attempted unsuccessfully to identify the genomic location of the dichloromethane-utilization genes (dcm) in Methylobac- terium sp. DM4.

Scholtz et al., J. Bacteriol., 170:12, 5698-5704 (1988) , found a second dichloromethane dehalogenase (a Group B dehalogenase) , distinct from that identified by Kohler- Stab et al . , in a methylotrophic bacterium denoted as "strain DM11".

Efforts to convert these various laboratory observa¬ tions to an industrial reality however have not met with any great success. The use of fluidized beds in the oxidative degradation of sewage on large scale is well known. Jeris et al . , U.S. Patent Nos. 3,846,289 and 3,956,129 for example describe a process and apparatus for denitrifying waste water in a fluidized bed of denitrifying biota attached to particles and providing a source of carbon to convert nitri¬ fied waste into nitrogen and carbon dioxide. The related U.S. Patent Nos. 4,009,098 and 4,009,105, also to Jeris, disclose a similar method and apparatus used, however, to remove what was called "biochemical oxygen demand" through oxidation of "biologically decomposable contaminants" to carbon dioxide and water.

Galli et al . , Cons. Recycling, 8: 91-100 (1985) exam- ined flow-through processes to degrade methylene chloride utilizing Hyphomicrobium sp. DM2 and several strains of Pseudomonas , DM1, DM2, DM3, DM4, DM5, DM5R (a streptomycin- resistant mutant), DM6, and DM10. Utilizing sand as a car¬ rier, the system was stable once maximum degradation capac- ity was reached but this was not the case when charcoal was employed as the carrier.

In Appl. Microbiol. Biotechnol., 27:206-213 (1987), Galli et al . attempted to use coal-derived charcoal as a support for a mixture of Hyphomicrobium sp. DM2, Methylo- bacterium sp. DM4, and Pseudomonas sp. DM5R in the biodegra¬ dation of methylene chloride but again the system was found to be instable in operation and sand again had to be used instead.

Speitel et al . , Jour. AWWA, 79:64-73 (1987) and Envi¬ ron. Sci. Technol. 23:68-74 (1989) describe a technique for extending the service life of activated carbon columns through in situ biological regeneration of sorption sites involving elimination through biodegradation of p-nitro- phenol, 2,4-dichlorophenol, and pentachlorophenol.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the apparatus employed to prepare the biodegradation matrix employed in the present invention;

Figure 2 is a schematic diagram of the bioreactor and associated equipment employed to degrade methylene chloride according to this invention; and

Figure 3 is a schematic diagram of an alternative arrangement of the bioreactor and associated equipment.

DETAILED DESCRIPTION

The present invention provides a process for the con¬ tinuous degradation of methylene chloride in aqueous waste streams. The process is so efficient that methylene chlor¬ ide is virtually undetectable in the effluent streams when measured by common analytical methods such as gas chromato- graphy. Thus aqueous waste streams having high levels of methylene chloride can be purified so that the level of methylene chloride in the effluent is below 5 parts per bil¬ lion.

In its broadest aspect, aqueous influents containing the methylene chloride to be degraded and dissolved oxygen are passed through a bioreactor containing a biodegradation matrix. The biodegradation matrix comprises a methyl¬ otrophic bacterium affixed by oxygen-promoted secondary cell attachment, probably polysaccharride-based, to particulate coal-derived charcoal. Under the appropriate conditions, as more fully described below, the methylene chloride is con¬ verted to carbon dioxide, hydrogen chloride, and biomass. The hydrogen chloride (in the form of hydrochloric acid) is neutralized with base. The bioreactor can be fully auto¬ mated and run continuously on the site at which the waste streams are being generated.

A critical operation in the present invention involves the preparation of the biodegradation matrix used to degrade the methylene chloride. Broadly, this operation involves isolating a methylotrophic bacteria which can utilize methylene chloride as its sole carbon and energy source but then culturing the microorganism in a growth medium in which methanol is the primary carbon and energy source. At about mid-point of logarithmic growth, the culture broth is brought into intimate contact with particulate activated coal-derived charcoal and oxygen then is introduced at least until substantial secondary cell attachment has occurred. The solid microorganism-charcoal biodegradation matrix from the reaction mixture then is separated for use in the methylene chloride bioreactor.

Alternatively, the microorganism can be immobilized on the particulate activated coal-derived charcoal in situ in the bioreactor. The process for degrading methylene chloride involves utilizing the biodegradation matrix in a bioreactor, dis¬ solving oxygen in the aqueous influent, passing the aqueous influent and dissolved oxygen through the biodegradation matrix, recycling a portion of the effluent from the biore¬ actor, and adding base to the liquid to neutralize hydrochloric acid -produced from the methylene chloride. Should the methylene chloride influent have a particularly high pH, acid can be added to it prior to processing.

The apparatus utilized will include a bioreactor opera¬ ble to both retain the biodegradation matrix and permit pas¬ sage of the aqueous liquid through the bioreactor over the biodegradation matrix. Means operable to meter nutrients into the aqueous liquid, means operable to introduce the aqueous liquid into the bioreactor, and means operable to remove the aqueous liquid after passage through the bio¬ reactor will be provided. Associated with the apparatus will be means operable to measure the pH of the aqueous liq¬ uid, and means operable to add base to neutralize hydrogen chloride. In addition, means operable to measure the con¬ centration of dissolved oxygen in the aqueous liquid, and means operable to dissolve oxygen in the aqueous liquid in response to these measurements will be provided.

The nature of the process, its components, and the apparatus are now addressed in detail.

I. Biodegradation Matrix Preparation A. Microorganism Isolation

The organism employed is a methylotrophic bacterium. Particularly useful are strains of HyphomiσroJiura, Methyl- obacterium, .and Pseudomonas. Of these Hyphomicrobium has proven to be most effective. _^_ "^''

Isolation of the microorganism generally is performed from soils having a history of exposure to halogenated hydrocarbons (the presence of halogenated hydrocarbons in the soil samples at the time of isolation, however, is not necessary) . The soil samples are incubated at 30°C with agitation in 30 L of a nitrate/mineral salts media contain¬ ing 50 mM phosphate buffer (pH 7.2) and having the following compositions:

Potassium nitrate 1.0 g

Magnesium sulfate (7H20) 1.0 g Calcium chloride (2H2O) 0.265 g

Trace elements 1.0 mL

Stock solution 1.0 mL

Water 1000.0 L

The trace element mix has the following compositions:

Ferrous sulfate (6H2O) 500 mg

Zinc sulfate (7H2O) 400 mg

Manganese sulfate (4H2O) 200 mg

Boric acid 15 mg Nickel chloride (6H₂O) 10 mg

Ethylenediaminetetraacetic acid 250 mg

Cobaltous chloride (6H2O) 50 mg

Cuprous chloride (2H2O) 5 mg

Water 1000 mL

The stock solution contains 5.0 g of ferric sodium ede- tate and 2.0 g of sodium molybdate in 1000 mL of water.

Methylene chloride is added to produce a concentration of 1000 ppm (calculated as if all were contained in the liq- uid phase) . Degradation of the methylene chloride can be readily monitored by gas chromatography. When the methylene chloride is exhausted, the samples are transferred to fresh media containing 50 mM of phosphate buffer (pH 7.2) and additional aliquots of methylene chloride are added.

After several transfers, the samples are plated on an ammonium/mineral salts media solidified with 2% noble agar containing 0.1% (w/v) carbon source (trimethylamine, methyl- formate, sodium formate, or succinate) . The ammonium/min¬ eral salts media has the following compositions:

Ammonium sulfate 0.66 g

Magnesium sulfate (7H2O) 1.0 g

Calcium chloride (2H2O) 0.265 g Trace elements 1.0 mL

Stock solution 1.0 mL

Water 1000.0 mL

The plates are maintained in an atmosphere of air and methylene chloride and colonies are picked. The picked colonies are further streaked on ammonium/mineral salts media with 20 mM of phosphate buffer (pH 7.2) and grown in an atmosphere of methylene chloride. Final colonies are picked from an ammonium/mineral salts media containing 0.1% formate and 50 mM of phosphate buffer (pH 7.2) and grown in an atmosphere of methylene chloride.

The isolated strains can be grown on ammonium/mineral salts media or nitrate/mineral salts media as described above. Cell stocks can be stored in either liquid nitrogen or a -70^βC freezer for subsequent use.

Unintentional contamination can be determined by micro¬ scopic examination. Contamination of flyphoinicroJiujn also can be detected by streaking a tryptic soy plate (on which this microorganism grows poorly) and incubating overnight, growth indicating contamination. ---^{^"^}

Several representative iϊyphoiπicroJium strains obtained following the foregoing procedures and suitable for use have been deposited with the American Type Culture Collection, Rockville, Maryland, under the Budapest Treaty as ATCC Nos. 55283, ATCC 55285, and ATCC 55308.

B. Fermentation

1. Inoculum Preparation

An inoculum is prepared by first autoclaving 1 L of ammonium/mineral salts media, after which 1% sterile methanol and 20 mM of phosphate buffer (pH 7.2) are added. A cryopreserved sample (4 mL) of Hyphomicrobium ATCC 55283 is added and the culture incubated at 30°C for 72 hours with stirring (250 rpm) to produce an ODggonm ^of between 0.5 and 1.0.

2. Fermentation While the following procedure is given for a 20 L fer¬ mentation vessel, direct scale-up to, for example, a 150 L fermenter (120 L working volume) presents no problems (and in fact can results in a shorter fermentation period) .

A fermentation vessel (20 L) fitted with a pH probe and dissolved oxygen probe is cleaned and rinsed and the follow¬ ing fermentation media added:

Ammonium sulfate 30.0 g

Magnesium sulfate (7H2O) 3.0 g

Calcium chloride (2H2O) 0.45 g Trace elements 30.0 mL

Stock solution 30.0 mL

Water 15.0 L

The pH probe is calibrated to pH 4 and 7 and the oxygen probe to 0% with sodium bisulfite and 100% with aeration prior to inoculating the vessel. The contents of the fer¬ mentation vessel are sterilized for 45 to 60 minutes at 121°C and 15 psi. Separately, 6N sulfuric acid and 28% ammonium hydroxide are sterilized. Following sterilization, aeration of the fermentation vessel is initiated at 0.8 vol. of air/vol. fermenter/min. (wm) . and the unit cooled to 30°C. Sterile 2M phosphate and filter-sterilized neat methanol are added to concentrations of 20 mM and 1% (v/v) , respectively. The fermentation vessel then is inoculated with the culture prepared above. Fermentation is continued for about 65 hours to the mid-point of logarithmic growth, by which time the ODggonm ^{of the} culture broth should be about 5 (2.25 g/L dry weight). Methanol concentration is monitored during the fermentation by gas chromatography and should be maintained between about 0.25% to about 5%, preferably between 0.5% and 1.0%, by addition of filter- sterilized neat methanol.

C. Immobilization

1. Charcoal Preparation

Two hundred twenty seven kilograms of granular acti¬ vated carbon (Atochem Inc. Cecarbon GAC 1240) is pretreated by cycling 240 L of solution of half-strength ammo¬ nium/mineral salts media (previously described) containing 1.0% methanol and 20 mM phosphate buffer (pH 7.2) for 24 hours to equilibrate the granular activated carbon with methanol and buffer, wet the surface, and eliminate carbon fines.

2. Cell Immobilization

The culture broth prepared as above is diluted with half-strength ammonium/mineral salts media containing 1.0% methanol as the sole carbon and energy source and 20 mM phosphate buffer (pH 7.2). A volume corresponding to approximately 20% of the volume of granular activated carbon to be treated is used.

Referring now to Figure 1, the pretreated granular activated carbon is disposed in immobilization chamber 10 fitted with air inlet 12, siphon break and liquid return 14, liquid inlet 16, and vent 28. The diluted culture broth is pumped at a rate of from about 1 to about 2 L/min from cul- ture reservoir 18 by pump 20 into immobilization chamber 10 where it is allowed to filter over the granular activated carbon and then return to culture reservoir 18. Siphon break 14 maintains the culture broth at a constant level within immobilization chamber 10 above the level of the pre- treated granular activated carbon. The pH of the circu¬ lating culture solution is monitored as by pH probe 22 and adjusted as needed by addition of 2N phosphoric acid from buffer reservoir 24, accomplished by pump 26 which in turn is controlled by pH probe 22 and associated control cir- cuitry (not shown) .

Air, preferably prefiltered, is introduced through inlet 12 at a constant rate, e. g. , about 2 L/min, and bub¬ bled through the granular activated carbon in counter-cur¬ rent fashion, escaping through vent 28.

In order to process a greater quantity of granular activated carbon, several immobilization chambers can be used in parallel with inlet 16 connected to a manifold inlet (not shown) .

By permitting this process to operate as described for a sufficient period of time, as for example 48 hours, cell growth and biopolymer production will take place on the sur¬ face of the granular activated carbon. Thus by introducing oxygen into the methanolic mixture, secondary cell attach¬ ment will occur between the microorganism and granular acti- vated carbon, thereby producing a biodegradation matrix hav¬ ing unique properties.

The biodegradation matrix is removed from the immobi- lization apparatus and can be stored at reduced tempera¬ tures, as for example about 4°C, until ready for use^' as described below.

Alternatively, the bacteria can be immobilized on the particulate activated carbon directly in the bioreactor. This in situ immobilization involves transport of cells and dry particulate activated carbon separately to the bioreac¬ tor site. The cells can be in the form of a cell broth, a cell paste, a lyophilized culture, or any other form which preserves the cells in a viable state. The particulate activated carbon is loaded into the reactor and the reactor then filled with water and startup nutrients are added. A period of time may be required to neutralize any alkalinity associated with the particulate activated carbon. The cells then are added to the reactor and total recycle is initi¬ ated, total recycle being the absence of liquid flowing out of the reactor while maintaining flow within the fluidized bed. The fluidization flow is set so as to maintain a com¬ pacted and non-fluidized bed of particulate activated car- bon. Typically this flow is about 25-30 gpm/m².

During total recycle, oxygen and methylene chloride are supplied to the bacteria. The oxygen can be conveniently supplied as dissolved oxygen through addition and dissolu¬ tion in the recycle line. An aqueous solution of methylene chloride is added by metering pump at the base of the biore¬ actor, the concentration of methylene chloride preferably being kept in the range of from about 50 ppm to about 1000 ppm. Methylene chloride addition in this fashion promotes bacterial growth and attachment only in the vicinity of the particulate activated carbon. To accelerate immobilization, bicarbonate can be added to the nutrient medium, typically at a concentration of about 0.25 g/1. Sources of bicarbon¬ ate include, but are not limited to, carbon dioxide, sodium bicarbonate, potassium bicarbonate, calcium carbonate, potassium carbonate, and sodium carbonate.

II. Bioreactor Operation

Referring to Figure 2, the influent waste water to be processed {designated as "I") , entering through input line 32, is pumped by feed pump 34 to bioreactor 30. It is to be appreciated that while the influent initially is the waste water feed, in the course of passage through the system it will be mixed with nutrients and return liquids, as dis¬ cussed in greater detail below. Hence while the composition of these materials will differ depending on the position within the system, the single term influent will be used for the sake of convenience.

Nutrients from nutrient reservoir 36 are metered into the influent with the aid of pump 38. The pH is measured at probe 40 and oxygen at probe 39, the liquid then entering bioreactor 30 through port 41. The oxygen content of the processed liquid is measured at second oxygen probe 43 and the pH at second pH probe 45. Recycle line 42 carries pro¬ cessed liquid back to gas trap 44. Base, typically sodium hydroxide, is metered in from base reservoir 46 by pump 48 in response to measurements by pH probes 40 and 45. Oxygen in turn is metered in at oxygen inlet 50 in response to oxy¬ gen probes 39 and 43. The recycled liquid, with pH adjusted and oxygen added, then is passed through gas trap 44 for removal of nondissolved gases and then returned to bioreac¬ tor 30.

The biodegradation matrix, prepared as described above, is disposed in bioreactor 30. The influent entering through port 41 passes upwards through the biodegradation matrix and then exits through either recycle line 42, as discussed above, or the effluent line {the exiting effluent being des¬ ignated "E"}. In the course of this passage, the biodegra¬ dation matrix converts the methylene chloride to carbon dioxide and hydrochloric acid^'. The carbon dioxide either is vented through vent 52, removed in gas trap 44 and vented through gas trap vent 47, or dissolved in the liquid as bicarbonate. The hydrochloric acid is neutralized by the base added from base reservoir 46.

Main fluidization pump 49 is operated so that working in conjunction with feed pump 34 the liquid passes through bioreactor 30 at a constant rate, typically advancing the liquid at a superficial linear velocity of from about 0.7 to about 1.5 cm/sec. The rate at which feed is pumped by pump 34 generally is inverse to the concentration of methylene chloride in the influent.

Because the entire objective is to remove methylene chloride, it is important to minimize bubbling in the bio- reactor which might entrap methylene chloride in gaseous form and not only prevent its degradation but also permit its escape into the atmosphere. Such bubbling is minimized by incorporation of gas trap 44 to trap undissolved gases.

While the apparatus can be operated in a single pass mode without recycling, particularly for low concentrations of methylene chloride, it generally is preferable to recycle as shown. In this way the recycled liquid can be used both to introduce maximum dissolved oxygen without stripping and to recirculate bicarbonate which acts as buffer, thereby assisting in control of the pH.

In the alternative configuration shown in Figure 3, the influent passes through filter 60 to remove any aprticulate matter. Base from base reservoir 46 is introduced through pump 48 downstream of gas trap 44. In addition, provisions ore made for the addition of acid from acid reservoir 56 through pump 58 in the event the incoming influent has a high pH. . Nutrients from nutrient reservoir 36 are metered into the influent with the^* aid of pump 38 downstream of probes 39 and 40 but before entrance into bioreactor 30.. On recycling, the oxygen which is metered in at oxygen inlet 50 can be mixed with the recycle liquid at mixer 54 prior to entrance into gas trap 44.

Typically granular activated carbon (apparent density of about 496 kg/cm³) is added to the reactor column such that with no flow through the column, the height of the car¬ bon bed is 2.25 m. A typical granular activated carbon will be a 12 by 40 mesh having an effective size of 0.6 mm. The preferred range of carbon size is from 0.3 to 2.0 mm. The superficial linear velocity of liquid through the column can range from 0.7 cm/sec (10.3 gpm/ft²) to 1.5 cm/sec (22.1 gpm/ft²) . In a reactor having an inside diameter of 20" (50.8 cm), this flow range corresponds to 22.5 to 48.2 gpm. The percent expansion in the bed of the nascent carbon ranges, for example, from 37% at 10.3 gpm/ft² and 18° C to 75% at 17 gpm/ft² and 18° C and is a function of tempera¬ ture. The optimum temperature to maintain activity of the bacteria ranges from 15 to 45° C. The fluidisation flow rate is adjusted in response to the actual temperature in order to maintain a constant percent bed expansion of the carbon.

A. Start-up Operation of the bioreactor unit requires a start-up period of several days. Initially the entire system is filled with water and the biodegradation matrix introduced into bioreactor 30. Base reservoir 46 is filled with diluted (about 5%) aqueous sodium hydroxide. Nutrient reservoir 36 is filled with high nutrient start-up feed (see below) .

An aqueous influent typically containing 1000 to 1500 ppm methylene chloride is introduced into the system through input line 32. The recycle ratio is approximately 10:1; i. e. , for a flow rate through bioreactor 30 of approximately 100 L/min. the input of untreated aqueous influent is about 10 L/min. This can vary, however, depending on the methyl- ene chloride content.

Since it is desirable to maintain the level of methyl¬ ene chloride entering at inlet 41 at a high but constant level consistent with the amount of dissolved oxygen, typi¬ cally from 100 to 150 ppm of methylene chloride, the recycle ratio is adjusted to dilute the influent, depending on its concentration, to this level.

High nutrient start-up feed is introduced from reser¬ voir 36. This is then metered into the influent stream by pump 38 to produce the following concentrations in the cir- culated liquid:

Component mg/L of influent

Potassium nitrate 500

Magnesium sulfate (7H2O) 50

Potassium phosphate monobasic 11.45 Potassium phosphate dibasic 2.75

Calcium chloride 2H2O) 7.5

Ferric sodium edetate 2.5

Sodium molybdate (2H2O) 10.0

Ferrous sulfate (7H2O) 0.25 Zinc sulfate (7H₂0) 0.2

Manganese sulfate (4H2O) 0.01

Boric acid 0.0075

Nickel chloride (6H₂0) 0.005

Ethylenediaminetetraacetic acid 0.125 Cobaltous chloride (6H2O) 0.025

Cuprous chloride (2H2O) 0.0025

pH is adjusted to about 6.5 by addition of base as needed from base reservoir 46. Oxygen is introduced through inlet 50 to maintain a level of approximately 15 ppm (dissolved). It will be appreciated that these values can and will be adjusted to accommodate the particular equipment and the composition of the influent.

Because the biodegradation matrix requires an induction period to become operational, the level of methylene chlo¬ ride in the influent initially entering at port 41 and that of the liquid exiting at recycle line 42 are not sig- nificantly different. After several days of continuous operation, however, an increase in chloride ion can be detected. Addition of base to maintain the pH, and intro¬ duction of oxygen, nutrient start-up feed, and aqueous influent should be continued during this time. By between one and two weeks of operation, the level of methylene chloride exiting at recycle line 42 should drop dramatically until the chlorine mass balance (based on methylene chloride entering and chloride ion formed) has reached approximately 1. The process then should be converted from start-up to steady state operation.

B. Steady State Operation

The process is converted to steady state conditions in the following manner. Oxygen input is increased and main¬ tained at a level of from 35 to 45 ppm. pH is held at a value of from about 5 to about 8.5. Nutrient start-up feed in nutrient reservoir 36 is replaced with a lower mineral nutrient running feed and fed by pump 38 to produce the fol¬ lowing concentration in the circulating liquid: Component ^"..... mg/L of influent

Potassium nitrate 10

Magnesium sulfate (7H2O) 1

Potassium phosphate monobasic 11.45 Potassium phosphate dibasic 2.75

Calcium chloride (2H2O) 0.15

Ferric sodium edetate 0.05

Sodium molybdate (2H₂O) 0.2

Ferrous sulfate (7H2O) 0.005 Zinc sulfate (7H₂0) 0.004

Manganese sulfate (4H2O) 0.0002

Boric acid 0.00015

Nickel chloride (6H₂0) 0.0001

Ethylenediaminetetraacetic acid 0.0025 Cobaltous chloride (6H2O) 0.0005

Cuprous chloride (2H2O) 0.00005.

The concentration of the two sources of phosphate, potassium phosphate monobasic and potassium phosphate di- basic, can be reduced to 0.229 mg/L and 0.055 mg/L, respec¬ tively, to reduce costs while still obtaining high effi¬ ciency.

During steady state operation, the pH as measured at pH probe 45 should be kept at a value of at least about 5, preferably 5.5 to 7.5, and oxygen as measured at oxygen probe 43 at a level of 2 ppm or higher. The pH at inlet 41 generally is held to between 6.5 to 8.5.

In a typical system, an aqueous waste stream obtained from a vapor scrubber and containing 2823 ppm of methylene chloride was fed into the apparatus at a rate of 10.8 L/min with a recycle ratio of 11.5. The superficial linear veloc¬ ity through the bioreactor ranged from 0.96 to 1.15 cm/sec. At the beginning of start-up, the concentration of methylene chloride exiting from the bioreactor was about 500 ppm as compared with a concentration of about 770 ppm entering the bioreactor. After 5 days, chloride ion began to increase and the level of methylene chloride exiting from the bio¬ reactor began to decrease. By day 11, the level of methyl- ene chloride exiting from the bioreactor was below the detection limit (5 ppm) of the on-site monitor. At day 15, the process was converted to steady state as described above and run continuously for over two months consistently pro¬ ducing high quality effluent containing less than 5 ppm methylene chloride (similar laboratory trickle bed systems have operated continuously for close to 12 months) . Moni¬ toring of effluent from pilot plant operations with more sensitive instrumentation have indicated methylene chloride levels often are below 5 ppb.

Claims

What is claimed is:

1. Process for the preparation of a biodegradation matrix operable to degrade methylene chloride on a continuous basis which comprises:

(i) culturing to about mid-log phase growth a methyl¬ otrophic bacteria capable of utilizing methylene chloride as its sole carbon and energy source in a growth medium in which methanol is the primary carbon and energy source;

(ii) bringing the bacteria into intimate contact with particulate activated coal-derived charcoal in the pres¬ ence of methanol; (Hi) introducing oxygen into the mixture at least until substantial secondary cell attachment has occurred; and

(iv) separating the solid microorganism-charcoal biodegradation matrix from the reaction mixture.

2. The process according to claim 1 wherein the concentra- tion of methanol during the growth of said methylotrophic bacteria is maintained at from about 0.25% to about 5% vol/vol.

3. The process according to claim 2 wherein the concentra- tion of methanol is maintained at from about 0.5% to about 1%.

4. The process according to claim 1 wherein said charcoal is treated with methanol and a source of phosphate anions prior to contact with said culture broth.

5. The process according to^" claim 1 wherein said bacteria are diluted prior to being brought into contact with said charcoal.

6. The process according to claim 5 wherein the volume of the diluted bacteria is from about 10% to about 50% of the volume of the charcoal.

7. The process according to claim 1 in which the meth- ylotrophic bacteria is a species of Hyphomicrobium.

8. The process according to claim 1 in which the methy¬ lotrophic bacteria is a species of Methylomonas .

9. A biologically pure culture of Hyphomicrobium Strain CEL 5002, ATCC 55283.

10. A biologically pure culture of tfypho_7.iσroJbium strain CEL 5016, ATCC 55285.

11. A biologically pure culture of Hyphomicrobium Strain CEL 5015, ATCC 55308.

12. A biodegradation matrix operable to degrade methylene chloride which comprises a methanol-grown methylotrophic bacteria affixed by oxygen-promoted secondary cell attachment to particulate^* activated coal-derived char¬ coal.

13. A biodegradation matrix according to claim 12 in which the methylotrophic bacteria is a strain of Hyphomicro¬ bium.

14. The process for degrading methylene chloride in an aque¬ ous influent which comprises

(i) providing a biodegradation matrix in a bioreactor, said matrix comprising a methylotrophic bacteria affixed by oxygen-promoted secondary cell attachment to particu¬ late activated coal-derived charcoal; (ii) dissolving oxygen in the aqueous influent; (Hi) passing the aqueous influent and dissolved oxygen through the biodegradation matrix;

(iv) recycling a portion of the effluent from the biore¬ actor; and (v) adding base to the recycled effluent to neutralize hydrochloric acid produced by the biodegradation matrix from the methylene chloride.

15. The process according to claim 14 in which the concen¬ tration of dissolved oxygen of each of the reactor influ- ent and reactor effluent is monitored and oxygen is added in response thereto.

16. The process according to claim 14 in which the pH of each of the reactor influent and reactor effluent is monitored and base is added in response thereto.

17. An apparatus for biologically reducing the concentration of methylene chloride in an aqueous liquid which com¬ prises:

(a) a bioreactor operable

(i) to retain a biodegradation matrix having a methylo¬ trophic bacteria affixed by oxygen-promoted sec¬ ondary cell attachment to particulate activated coal-derived charcoal, and

(H) to permit passage of the aqueous liquid through the bioreactor and over said biodegradation matrix for conversion of the methylene chloride to at least carbon dioxide and hydrogen chloride;

(J ) means operable to introduce said aqueous liquid into said bioreactor;

σ) means operable to meter nutrients into said aqueous liquid;

(d) means operable to measure the pH of said aqueous liq¬ uid;

(e) means operable to add base to said aqueous liquid to compensate for decrease in said pH measurements through formation of hydrogen chloride;

(f) means operable to measure the concentration of dis¬ solved oxygen to in aqueous liquid;

(g) means operable to dissolve oxygen in said aqueous liquid in response to said oxygen measurements; and

(h) means operable to remove said aqueous liquid after passage through said bioreactor.

18. The apparatus according to claim 17 including means operable to recycle a portion of said aqueous liquid after passage through said bioreactor for combination with untreated aqueous liquid to be introduced into said bioreactor.

19. The apparatus according to claim 18 wherein said oxygen dissolving means are associated with said recycling means.

20. The apparatus according to claim 18 wherein said base addition means are associated with said recycling means.

21. The apparatus according to claim 18 wherein said oxygen dissolving means and base addition means are associated with said recycling means, said pH measuring means and said dissolved oxygen measuring means are operable to monitor aqueous liquid entering the bioreactor, and sec- ond pH measuring means and second dissolved oxygen mea¬ suring means are operable to monitor aqueous liquid in said bioreactor proximate to said recycle means.