US3586605A

US3586605A - Method for controlling hydrocarbon fermentations and apparatus therefor

Info

Publication number: US3586605A
Application number: US737991A
Authority: US
Inventors: Peter Hosler
Original assignee: Sun Oil Co
Current assignee: Sunoco Inc
Priority date: 1968-06-18
Filing date: 1968-06-18
Publication date: 1971-06-22
Anticipated expiration: 1988-06-22

Abstract

THE CONCENTRATION OF A MODERATELY VOLATILE HYDROCARBON SUBSTRATE IN A FERMENTATION MEDIUM CAN BE READILY DETERMINED AND SAID CONCENTRATION MAINTAINED AT A PREDETERMINED LEVEL BY MONITORING THE VAPOR CONCENTRATION OF SAID HYDROCARBON IN THE FERMENTATION EXHAUST GASES AND CONTROLLING THE INTRODUCTION OF ADDITIONAL SUBSTRATE INTO THE FERMENTATION MEDIUM BASED ON THE AMOUNT OF SAID VAPOR CONTENT. THIS CAN ADVANTAGEOUSLY BE CARRIED OUT IN A CONTINUOUS MANNER BY AUTOMATED INSTRUMENTATION.

Description

June 2 1971 P. HOSLER 3,586,605

METHOD FOR CONTROLLING HYDROCARBON FERMENTATIONS AND APPARATUS THEREFOR Filed June 18, 1968 MONITOR ANALYZER,

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PETER HOSLER JOMJJK ATTORNEY United States Patent O1 fice Patented June 22, 1971 US. Cl. 195-28 6 Claims ABSTRACT OF THE DISCLOSURE The concentration of a moderately volatile hydrocarbon substrate in a fermentation medium can be readily determined and said concentration maintained at a predetermined level by monitoring the vapor concentration of said hydrocarbon in the fermentation exhaust gases and controlling the introduction of additional substrate into the fermentation medium based on the amount of said vapor content. This can advantageously be carried out in a continuous manner by automated instrumentation.

BACKGROUND OF THE INVENTION This invention relates to an improved method for controlling the concentration of hydrocarbon substrates in fermentation media where said hydrocarbons are being oxidized by microorganisms to form oxygenated hydrocarbon derivatives. It also relates to an apparatus for achieving this purpose.

The microbiological oxidation of hydrocarbons to form oxidative products thereof has been investigated and reported in numerous recent literature and patent references. Not as well known or understood, however, is the need for controlling the concentration of the hydrocarbon substrate in the fermentation medium in order to achieve improved yields of the desired oxidation products. Thus, in US. Pat. No. 3,458,399, filed by the present applicant on Aug. 31, 1966, there is taught the desirability of controlling the rate of addition of the hydrocarbon feed so that the amount thereof in the dispersion in excess of the amount absorbed by the microorganism cells does not exceed the limit of solubility of the hydrocarbon in the aqueous nutrient medium. This thus-desired control, as taught by this earlier application, was achieved by taking samples of the fermentation medium from time to time, testing them to determine the concentration of hydrocarbon substrate in the aqueous phase of the medium and regulating the introduction of additional feed accordingly. While the effect of this sampling and control was to increase the yield of oxidative product, nevertheless complex testing proceduress involving centrifuging the samples, extracting them with suitable solvents and analyzing the extract by UV spectroscopy were required at each sampling interval. Despite these elaborate methods, such steps were only partially successful in achieveing the desired control.

Therefore, if control of the hydrocarbon concentration were ever to be achieved on a practical basis, it became evident that the known sampling methods would have to be improved upon in order to provide more simplified and accurate methods for obtaining this desired objective.

Accordingly, it is an object of this invention to provide an improved method, and apparatus, for closely and accurately measuring and controlling the concentration of a hydrocarbon substrate at a predetermined level in a fermentation medium where said hydrocarbon is undergoing microbiological oxidation. This and other objects of the present invention will be apparent from the following description.

SUMMARY OF THE INVENTION In accordance with the present invention it has now been found that the concentration of a moderately volatile hydrocarbon substrate in an aqueous fermentation medium can be readily determined and said concentration closely controlled and maintained at a predetermined level by:

(1) Establishing under aerobic fermentation conditions an aqueous fermentation medium comprising a hydrocarbon-oxidizing microorganism and a predetermined amount of a liquid hydrocarbon substrate dispersed in an aqueous nutrient medium;

(2) Sampling the exhaust gas from said fermentation to determine the vapor content of said hydrocarbon in said exhaust gas; and

(3) Controlling the introduction of additional hydrocarbon substrate into the fermentation medium in dependence upon the vapor content of said hydrocarbon in the exhaust gas in order to maintain said predetermined concentration.

DESCRIPTION OF THE DRAWING The figure is a schematic view of a fermentor in combination with hydrocarbon vapor analyzer which actuates a feed pump for regulating the rate of introduction of hydrocarbon feedstuif into the fermentor.

DESCRIPTION OF THE PREFERRED EMBODIMENTS As taught in US. Pat. No. 3,458,399 (supra), in order to obtain maximum yields of oxygenated hydrocarbon product from the fermentation of hydrocarbon feedstuifs, the introduction of the feed must be carefully controlled to maintain its concentration in the aqueous phase below that corresponding to the limit of its solubility in the nutrient medium; the amount of hydrocarbon absorbed by the cells of the microorganism is not counted for purpose of this determination. This solubility limit will depend mainly on the particular hydrocarbon used and the fermentation temperature employed. Hydrocarbon solubility limits in the nutrient medium usually are less than 200 ppm. and often may be less than p.p.m. In some cases it may be best to maintain a quite low hydrocarbon concentration, e.g., 5-20 ppm. as some microorganisms can be sensitive to and adversely affected by a higher concentration even though it be substantially below this solubility limit. Below a concentration of about 5 ppm, however, the rate of fermentation is generally undesirably slow.

Inv oxidizing alkylbenzenes of the C C range, for example, best results are generally obtained when the unconsumed hydrocarbon concentration is maintained at a level of about 5150 ppm. in the aqueous nutrient phase. For other types of substrates, and for any given microorganism, this optimum level can conveniently be determined experimentally by varying the hydrocarbon concentration levels and observing both the product yield and rate at which it is produced, as well as monitoring the oxygen and carbon dioxide levels in the exhaust gas of the fermentor. A. decrease in carbon dioxide and an increase in oxygen, for example, is indicative of an oversupply of hydrocarbon feed. The reverse is true when the feed supply is inadequate.

Once the Optimum concentration for any given combination of substrate and microorganism had been determined, it had been the practice formerly to maintain this concentration by taking samples of the fermentation broth at given intervals, separating the cells from those samples by centrifugation or filtration, extracting the unconsumed hydrocarbon, determining the amount extracted, and adding additional feed in accordance with this determination.

It has now been found, however, in accordance with the present invention, that these time-consuming and laborious steps can be avoided, and the concentration of hydrocarbon in the fermentation broth controlled much more closely and accurately, by directly measuring the vapor content of the hydrocarbon feedstuif in the exhaust gases of the fermentor without having to resort to taking samples of the fermentation broth. That is to say, it has been found that the concentration of hydrocarbon in the exhaust gas is directly proportional to the concentration of hydrocarbon in the aqueous fermentation. There thus exists an equilibrium distribution of the hydrocarbon between the aqueous fermentation and the fermentation exhaust gas which conveniently permits the monitoring of the hydrocarbon vapor content of the exhaust gas by appropriate measuring devices. Preferably, continuous monitoring by automatic instruments is provided Where the signal from the exhaust gas analyzer may then be used to determine the hydrocarbon feed rate, desirably by actuating a feed mechanism.

While applicant does not wish to be bound by any particular theory, the direct correlation that has been found to exist between the vapor content of the hydrocarbon in the exhaust gas and its concentration in the aqueous phase of the fermentation broth is apparently based on the principle that the partial vapor pressure of any volatile constitutent of a solution is equal to the vapor pressure of the pure constituent multiplied by the mol fraction of that constituent in solution. Based on this principle the concentration in the aqueous phase can readily be determined from the vapor content and the rate of feed to the fermentation broth conveniently controlled and maintained by appropriate devices.

The limits of the relationship between the vapor phase content and the aqueous phase content of the hydrocarbon are the solubility of hydrocarbon in water, and the saturation of the exhaust air stream, as influenced by the partial pressure of the hydrocarbon at fermentation temperature. In the case of a fermentation of xylene, for example, where the xylene is fermented at 30 C., the distribution has been found to follow the following relationship: 1 mg. xylene/liter of water=0.84 mg. xylene/liter of exhaust as. g The limit of the above relationship is approximately 37 mg. of xylene per liter of exhaust gas, which is the saturation point.

While various modes of operation will be apparent to those skilled in the art based on the foregoing description of the invention, the figure has been included to illustrate one typical method of operation which is applicable to different combinations of microorganisms and hydrocarbon substrates.

In the figure there is shown fermentor containing culture medium 11 which has been inoculated with a suitable microorganism. The fermentor is fitted with agitator 12 driven by motor 13. The agitator may be one or more in number depending upon the degree of agitation and aeration desired for any given fermentation. Fermentor 10 is also fitted with an aerator 14 through which sterile air can be bubbled from pipe 15. Optionally the fermentor 4 may be fitted with a pH measuring device which actuates a container of alkali, in order to maintain a given pH level.

At the top of the fermentor 10 the gases are exhausted with pressure from the pressurized fermentor through pipe 16 fitted with a back-pressure regulating valve 17. These gases are then passed along pipe 16 to which are attached oxygen monitor 18, carbon dioxide monitor 19, and a hydrocarbon analyzer 20. The remaining unsampled gases are then vented from pipe 16.

The oxygen and carbon dioxide monitors, which may be of any conventional construction, are employed for the purpose described earlier of measuring the respiratory activity of the cells, which in turn is a measure of their product-forming ability. While

monitors

18 and 19 are not essential to the performance of this invention, they do simplify the overall control of the fermentation system, and particularly the start-up procedures.

The electrical signal from hydrocarbon analyzer 20 is used to activate pneumatic converter 21, which may be of any conventional design. The regulated air output of this converter is, in turn, used to activate pneumatic valve 22. The hydrocarbon feed, in a pressurized vessel 23, is thus supplied through pipe 24 to the fermentor 10 in a proportional mode of operation to maintain a predetermined concentration of hydrocarbon substrate dispersed in the aqueous medium. If it is desired to maintain asepsis of operation, the hydrocarbon can be sterlized by passing it through a steam-jacketed heat exchanger 25. The start-up of this operation is ordinarily done manually to avoid initial overshoot until the overall system has reached equilibrium by responding to the vapor content of the exhaust gas.

As an alternative to proportional control the hydrocarbon feedstuif can be pumped to the fermentor using a simple relay to provide ofiF-on control of a pump attached to vessel 23, which relay is activated by the output of the hydrocarbon analyzer. This mode of control can be quite effective if care is taken to set the pumping rate only slightly in excess of required feed rate, so that excessive over-supply of hydrocarbon is avoided.

The hydrocarbon analyzer may be one of several different types which are commercially available. Thus, for example, a unit which measures ultra-violet light at the particular wavelength of the particular hydrocarbon feedstuff is suitable for purposes of this invention, especially if the hydrocarbon feed is an aromatic compound. Alternatively, a total hydrocarbon analyzer with a flame ionization detector may be employed instead, particularly if the hydrocarbon is a straight chain alkane.

The present invention is applicable to a wide variety of processes involving the fermentation of hydrocarbonoxidizing microorganisms in combination with various hydrocarbon substrates to produce many different types of oxygenated hydrocarbon products. Microorganisms wh1ch may be employed in these fermentations include bacteria, yeasts, molds and actino mycetes, and particularly such genera as Micrococcus. Corynebacterium, Nocardia, Pseudomanas, Mycobacterium, Streptomyces, Aspergillus, and Acetobacter.

The hydrocarbon substrates include any moderately volatile, normally liquid hydrocarbon having from 5 to 12 carbon atoms, and particularly the aromatic hydrocarbons such as the C C benzenoid hydrocarbons. Thus, for example, such hydrocarbons as benzene, toluene, xylene, hexane, octane, decane, dodecane and biphenyl or the like may suitably be employed in this process.

Among the types of oxidations wherein this invention has particular utility are in the oxidations of alkyl groups of alkylbenzenes as described in US. Pat. 3,057,784. The invention is likewise applicable to the oxidation of alkylnaphthalenes such as l-methylnaphthalene, l-ethylnaphthalene and the like, as well as to normally liquid alkanes of the C -C range, particularly n-parafiins and cycloparafiins.

Except for the control of the introduction of the liquid hydrocarbon substrate into the fermentation medium, the fermentation may be carried out in a conventional fashion wherein a nutrient medium is introduced into a fermentor provided with means for aeration and agitation, the medium inoculated with a suitable microorganism, and the fermentation is then effected by continually adding the hydrocarbon feed at a proper rate while maintaining fermentation conditions. Generally a temperature of from -40 C. is suitable, and the pH should be maintained in the range of 4-9, and preferably 6-8. The hydro carbon substrate may be added from the beginning, or else the cells can be grown on another substrate and then the hydrocarbon feed added.

After maximum accumulation of the oxygenated product, the fermentation broth can be treated in any suitable manner to recover the product. For example, the product can be extracted directly from the Whole broth by means of a suitable solvent, or else the cells may be first removed by centrifugation or filtration, followed by processing of the clear broth.

The nutrient medium used in the process should contain sources of available nitrogen, phosphorous, sulfur and magnesium and may contain various trace elements and vitamins as conventionally employed or as required by the particular microorganism being used. Mineral salts customarily used for supplying such elements in biological fermentations can be employed. Examples of suitable nitrogen sources are ammonium salts such as (NH SO or NH Cl, nitrate salts such as NH NO or NaNO urea, soybean meal and other organic nitrogen sources. The following illustrates a suitable mineral salt composition for the present purpose:

Conc., g./1. of H 0 0.2 Na CO 0.1 CaCl 2H O 0.01 MnSO -H O 0.02 FeSO 7H O 0.005 Na HPO 3 .0 KH PO 2.0 Urea 2.0

This mineral salt composition normally would have a pH of about 7.1. When it is desired to carry out the fermentation at a pH below 7, the amount of KH PO relative toNa HPQ, can be increased to reduce the pH to a lower 4 Nocardia corallina ATCC No. 19,070 was used to prepare dimethyl muconic acid (DMMA) from p-xylene in a 760 liter fermentor of the type shown in FIG. 1, together with an automatic pH monitoring system. A mineral salt solution of the following composition was used as the aqueous nutrient medium:

Conc., g./l. of H 0 MgSO -7H O 0.2 N11200:; 0.1 CaCl 2H O 0.01 MnSO -H O 0.02 FeSO -7H O 0.005 Na HPO 3.0 KH PO 2.0 Urea 2.0

520 liters of the solution were charged and sterilized and the mixture was inoculated with the organism. The mixture was stirred vigorously at 30 C. while being aerated at a constant rate of 2 c.f.m. Normal parafiins Were used as a growth substrate and were added at the rate of ml./hr. Also the pH level was automatically held at 6.8 during the run by addition of aqueous caustic soda using a pH controller. The exhaust gas was monitored by an ultraviolet absorption instrument, and the control apparatus was set to maintain 7 mg. xylene per liter of exhaust gas. At the end of the fermentation at about 50 hours analysis of the whole broth showed that it contained about 15.4 gm. of DMMA per liter.

EXAMPLE II In this run Nocardia: salmonicolor ATCC 19,149 was used to oxidize p-xylene to dihydroxy p-toluic acid (DHPT) and p-toluic acid (PTA). The procedure and apparatus were the same as in Example I. 34 liters of a mineral salt mixture were sterilized in a 60 liter fermentor provided with aeration and agitation. The mixture was inoculated with the organism and stirred vigorously at 30 C. while being aerated at a constant rate of 9 liters per minute. Normal paraflins were used as a growth substrate and were added at the rate of 7 ml./hour. At 12 hours the cell mass had increased to approximately 3 gm. dry weight per liter. At this time the pH was raised from about 7 to pH 8, and the xylene maintained at 20 gm. per liter of exhaust gas. After 20 hours of the controlled xylene addition the fermentation broth was analyzed and found to contain 4.2 gm. of DHPT and 5.3 gm. of PTA per liter.

EXAMPLE III The culture Nocardia salmonicolor ATCC 19,149 was grown on benzene as a growth carbon source to provide quantities of cell mass of the culture. 34 liters of a mineral salt mixture were sterilized in a 60 liter fermentor provided with aeration and agitation. The mixture was inoculated with the organism and stirred vigorously at 30 C. while being aerated at a constant rate of 6 liters per minute. Benzene was added to the system described above in Example I to maintain 12 mg. benzene per liter of exhaust gas. After 25 hours of operation growth was determined by centrifugation at 3600 rpm. A packed cell volume of 3% was obtained.

What is claimed is:

1. A process for determining the concentration, in an aqueous fermentation medium, of a hydrocarbon having from 5 to 12 carbon atoms, and maintaining said concentration at a predetermined level which comprises (a) establishing under aerobic fermentation conditions an aqueous fermentation medium;

(b) sampling the exhaust gas from said fermentation to determine the vapor content of said hydrocarbon in said exhaust gas; and

(c) controlling the introduction of additional hydrocarbons into the fermentation medium in dependence upon the vapor content of said hydrocarbon in said exhaust gas in order to maintain said predetermined concentration.

2. The process according to claim 1 wherein the sampling and controlling are carried out in a continuous manner.

3. The process according to claim 1 wherein the sampling and controlling are carried out automatically.

4. The process according to claim 1 in which the hydrocarbon is an alkylaromatic compound having from 5 to 12 carbon atoms.

5. The process according to claim 1 in which the hydrocarbon is a normally liquid alkane having from 5 to 10 carbon atoms.

6. A fermentation system for the controlled fermentation of hydrocarbons having from 5 to 12 carbon atoms wherein said hydrocarbons are maintained in a fermentation medium at predetermined concentrations which comprises, in combination, a fermentor containing a suitable fermentation medium, said fermentor being equipped with means for introducing controlled amounts of hydrocarbons into said fermentation medium and means for aeration and agitation of said medium, means for removing exhaust gases from the upper end of said fermentor, a hydrocarbon analyzer adapted to be receptive to the hydrocarbon vapor content of said exhaust gases, said analyzer being constructed and arranged to produce an output corresponding to said hydrocarbon vapor content, and means responsive to said output for controlling the output of the hydrocarbon-introducing means in amounts proportional to the output of said analyzer in order to maintain said hydrocarbons at predetermined concentration in the fermentation medium.

References Cited UNITED STATES PATENTS 3,010,881 11/1961 Markhof 195133X 3,383,289 5/1968 Raymond et al 195-28 3,384,553 5/1968 Caslavsky et a1. 19594X 10 A. LOUIS MONACELL, Primary Examiner S. RAND, Assistant Examiner US. Cl. X.R.