SPECIFICATIONEnzymatic treatment of organic substances and biomassThe invention relates to the conversion of organic compounds, suspended or dissolved in liquids, into stable reaction products, in particular, into carbon dioxide and water, through the addition of enzymes. The process according to the invention is particularly suitable for stabilising sewage sludge and slurries, as well as for treating organically burdened or phosphate-containing waste waters.
The invention also relates to the stimulation of the basal metabolism of micro-organism cultures used in the production of antibiotics, enzymes and organic acids.
In practice, organic media such as sewage sludges and slurries, are treated successfully by means of anaerobic, aerobic and enzymatic stabilisation methods. The enzymatic processes, introduced only during the past few years, enable a marked reduction in the stabilisation times of hitherto several days (5 to 70 days) of the anaerobic and aerobic methods to just a few hours, albeit at a much higher energy consumption, because the decomposition processes in the sludge or slurry, catalysed by the enzyme products, can take place successfully only at higher temperatures (30 to 60 C).
In addition, a considerable addition of extraneous enzymes is necessary.
Moreover, due to the inherent metabolism of the micro-organisms, which multiply considerably during the initial phase and also during the enzymatic stabilisation, many undesira- ble effects, such as impaired concentration and hydro-extraction capacity, are brought about, so that enzymatically stabilised sludges and slurries generally display inferior hydro-extraction properties compared with the conventional methods of stabilised sludges and slurries.
The treatment of organically burdened waste waters usually takes place according to the known activated sludge process. A major disadvantage of this process is the considerable re-formation of biomasses. To date, this disadvantage can be countered only by extending the aeration time.
Highly concentrated organic industrial effluents are usually treated by means of particularly highly burdened biological methods ("intensive biology"). In this so-called "intensive biological" treatment, a high percentage (often above 50%) of the organic substance to be decomposed, is converted into biomass, which has to be removed from the system as excess sludge, otherwise the efficiency of the process is low. However, the removal of the excess biomass in the "intensive biological" methods is always very problematical, because the micro-organisms grow only as individual cells or as very small complexes which seldom precipitate in the normal size post-settling basins.
Communal effluents generally have a high phosphate content. With the known biological elimination methods, the phosphate is released by anaerobic treatment, which requires a considerable reaction volume.
The large scale microbial production of antibiotics, enzymes and organic acids takes place submersed in aerated and agitated reactors (fermentators), usually by batchwise procedures.
For the successful completion of a fermentation, several pre-cultures are necessary to inoculate the production tank (main fermentation) together with sterile conditions.
In the case of batchwise fermentation, the micro-organisms (bacteria or fungi) usually grow until such time that an essential component of the nutrient medium becomes a limiting factor. The micro-organism culture then changes from the exponential growth phase into a stationary one.
During the fermentation process, the culture conditions are subject to permanent changes, thus leading to a change in the physiological condition of the micro-organism cells. As a rule, the desired product formation (antibiotics, enzymes or organic acids) are bound to a certain physiological state of the micro-organism during the fermentation. It is not possible to maintain this condition for any length of time during batchwise fermentation. Consequently, the enzymes are often discharged at the end of the logarithmic phase and continues over a more or less long period of time in the stationary phase. For example, antibiotics are often not formed in the culture solutions during the period of greatest growth, but rather only when the product begins to age, i.e. during the autolysis stage.Even the formation of organic acids, e.g. citric acid, commences only on completion of the producer growth. To achieve this, the culture of the citric acid agent takes place in the nutrient with suboptimum phosphate quantities.
The duration of fermentation is an experience value, and is to be viewed mainly under economic aspects. In order that fermentation is stopped at an optimum time, it is very important to monitor the fermentation process accurately. As a rule, a normal fermentation lasts 2 to 7 days. Following decomposition of the product-containing culture medium from the fermentors, and following thorough cleaning and maintenance, these are prepared for a renewed fermentation (filling with nutrient, sterilisation of the whole system, inoculation with pre-culture from the inoculation tanks).
Batchwise fermentation is of great industrial significance. At present, this is used only during the production of biomasses and fermentation products and for the conversion of organic products into gaseous reaction products and water. It has not been possible hitherto, to achieve such reaction conditions in a continuous process, such that the desired products are liberated from the cells of the microorganisms. Conversion of organic substances, e.g. waste products into CO2 and water, takes place only with very protracted aeration times, to be satisfactory. With economically acceptable aeration times, there is a high level of reformation of biomasses (anabolism), for which it is not always possible to find a satisfactory use. To date, no methods have been known to reduce the anabolism or constructive metabolism in favour of basal metabolism.The continuous process also sets high demands on the stability of the production bases during the production of certain substances, because with protracted continuous cultures, there is frequently a degeneration phenomena in the high-output bases.
The object of the invention is to achieve a high decomposition rate with small quantities of extraneous enzymes, to improve the hydroextraction properties of the enzymatically stabilised sludges and slurries, and to minimise the (thermal) energy necessary for the enzymatic stabilisation process, such that by comparison with the conventional stabilisation methods (putrefaction/fermentation, aerobic stabilisation), there are no longer any disadvantages with regard to hydroextraction behaviour and energy consumption.
Moreover, the production of excess sludge is to be reduced in activated sludge installations, and in particular, the biomass concentration is to be reduced in the "intensive biological" methods, and a sludge is to be produced which lends itself to sedimentation.
With the biological phosphate-elimination, the P-release from the activated sludge is to be greatly accelerated. Moreover, for the production of antibiotics, enzymes and organic acids, a continuous procedure is to be evolved for the micro-organism cultures.
The object of the invention is to stimulate the basal metabolism of micro-organism cultures in such a manner that there is a material loss of the organisms in the nutrient-containing medium, and consequently the growth process of the cultures is interrupted.
The object is achieved by the characteristics expounded in the patent claim.
The procedure of the method is as follows:The chelate-former brings about a weakening in the structure of the micro-organism cell walls, such that under corresponding stress, they can be dissolved. In the second reaction stage, they can be dissolved. In the second reaction stage, there is formed a medium which initially promotes the basal metabolism, and then promotes the autolysis or sporulation of the micro-organisms which stem from the first reaction stage. Apart from residues of the nutrient components, this contains essentially the low-molecular compounds (sugar, amino acids, fatty acids, alcohols, etc.) and the already autolysed micro-organisms or their fragments themselves, and the formed spores, all being easily realisable microbially and stemming from the enzymatic hydrolysis of the organic macro-molecules of the nutrients.
With the addition of the micro-organism culture, pre-treated with the chelate-formers, into this medium, there is a temperature shock due to the temperature difference of at least 10 K, as the result of which the basal metabolism of the micro-organisms is stimulated spontaneously, and the low molecular hydrolysis products contained in the medium, as well as the low-molecular compounds, are partially respirated from the cell disintegrations.
Because of the greater demands on the basal metabolism than on the constructive metabolism or anabolism, the micro-organisms in the reaction medium ultimately collapse due to material loss, i.e. they starve and convert to self-dissolution (autolysis state) or ultimately spored.
In the treament of organic waste products, the intro-cellular enzyme systems, released from the autolysed micro-organisms, give rise to an intensification of the hydrolysis of the organic macro-molecules. Consequently, considerable quantities of extraneous enzymes are saved. The microbial respiration of the lowmolecular compounds (especially, the hydrophilic colloids), originating mainly from the enzymatic degradation of the organic macromolecules and from the cell breakdown, improves the concentration and hydroextraction behaviour of the treated organic medium to a high degree. At the same time, the organic burdening of the incoming slurries is greatly reduced, thereby making possible a troublefree utilization of the slurries.
During the bio-technological production of certain substances, the product formation is initiated even during the stage of increased basal metabolism or only with the onset of the autolysis stage, depending on the product (antibiotics, enzymes, organic acids) and depending on the producer (type, origin). From the second reaction stage, the fermentation medium is released continuously, and can be delivered to the preparation stage. The antiorganism conditions prevailing in the fermentation medium of the main fermentor, virtually completely eliminates external infection, so that there is no need for a sterile fermentation in this phase.
The following reactions occur during the biological phosphate elimination: During aeration phase, the micro-organisms of the activated sludge absorb phosphate from the waste water to excess and form a granular complex of phosphate, PNA, proteins and lipides, referred to as "volutin". Through the addition of enzymes, the basal metabolism of the micro-organisms is stimulated, and the stored volutin is released.
This action is further intensified by the tem perature difference between the activated sludge and the reactor contents. Besides the release of the bound phosphate, effluent constituents are also converted into organic acids which are suitable as nutrients for micro-organisms, and which can store large quantities of phosphate in excess. The said processes are further accelerated by the treatment with the chelate-former, which gives rise to a breakdown of the micro-organism cell walls.
The advantage of the process relative to the anaerobic treatment is seen in the very short treatment time.
The invention will now be described with reference to eight examples:Example 1: Treatment of sewage sludge-- Variant 1;Example 2: Treatment of sewage sludge-- Variant 2;Example 3: Treatment of liquid manures (slurries);Example 4: Treatment of waste water from a slaughterhouse;Example 5: Treatment of waste water;Example 6: Production of penicillin;Example 7: (Fig. 1) Biological P-elimination without sludge water segregation;Example 8: (Fig. 2) Biological P-elimination with sludge water segregation.
Example 1To 15 m3 activated sludge with a solids content of 4% are added 30 g of a mixture of di-ammonium- and tri-ammonium salt of nitrile tri-acetic acid, previously dissolved in about 10 litres tap water. The addition is made with stirring. After 30 minutes, the thus pretreated sludge is fed batchwise to the reactor at intervals of 15 minutes; the reactor already contains sludge pre-heated to 550C.
The reaction time in the reactor is 3 hours.
For every cubic metre pretreated sludge added, 60 g of a complex enzyme product, containing ss-glucanase, amylase, proteases and lipases, pre-dissolved in about 100 times the volume of tap water, are added constantly. The sludge in the reactor is constantly circulated by means of a pump, and through the introduction of extraneous energy (e.g.
steam), the necessary reaction temperature is maintained.
The stabilised sludge passing through the reactor possesses good precipitation properties. In the following thickener, about 60% of the starting sludge appears as sludge water following a dwell time of about 2 hours, so that the volume of the stabilised sludge reduces to 40% of the starting volume. The concentrated, stabilised sludge is then piped to hydroextraction sites or artificially hydroextracted, while the sludge water is fed to the biological section of the sewage works for harmless disposal.
Example 2To a reactor, which already contains sludge preheated to 50 C, are added simultaneously but not mixed, a mixture of di-ammonium- and tri-ammonium salt of nitrile tri-acetic acid, predissolved in tap water, and a complex enzyme preparation containing ss-glucanases, amylases, proteases and lipases, pre-dissolved in about 100 fold volume of tap water, such that the introduction of the chelate-former can be made into the sludge infeed line or directly into the reactor.
Example 310 m3 liquid manure/slurry with 6% solids are reacted with 45 g tri-ammonium salt of nitrile tri-acetic acid, pre-dissolved in about 15 litres tap water, and after thorough mixing, stored for 1 to 3 hours. Thereafter, the slurry, containing the chelate-former is fed batchwise at intervals of 5 minutes, to the stabilisation reactor which already contains slurry heated to 33 C. The reaction time in the reactor is 8 hours. For each cubic metre chelate-containing slurry, are constantly added 90 g of a complex enzyme preparation, containing fi-gluca- nases, amylases, proteases and lipases, predissolved in about a 100 fold quantity of tap water. By means of an intensive aerator, the slurry is circulated in the reactor and supplied with oxygen.The slurry temperature rises to about 400C and then remains fairly constant.
As indicated in Example 1, the stabilised substance (slurry in this case), is concentrated in a thickener, and can then be hydroextracted artificially or on slurry beds. The volume of the thus stabilised slurry decreases by about 40 to 50% compared with untreated material in the thickener. The resultant sludge water can be piped to harmless utilisation.
Example 450 m3 organic, highly contaminated waste water from a slaughterhouse complex with a BSB, of about 7000 mg/1 and with a high protein and fat content, are mixed with 10 g of tri-ammomium salt of nitrile tri-acetic acid, pre-dissolved in about 5 litres tap water. After one hour, the organically highly contaminated effluent, containing the chelate-former, is fed batchwise, at intervals of 20 minutes, to the intensive biological initial stage, which already contains effluent pre-heated to 33 C. After a reaction time of 5 hours and in the presence of ss-glucanases, proteases and lipases, enzymatic hydrolysis takes place with microbial decomposition of the organic main contaminant of the effluent to be treated.For every cubic metre waste water added, are constantly added 10 g ss-glucanase, proteases and li- pases, pre-dissolved in 500 times the volume of tap water. Aeration of the intensive biological initial stage is effected with a high-power fan. Since the resultant heat of oxidation is not sufficient to maintain the necessary reaction temperature of 33 C, the reaction medium is additionally heated with steam.
The enzymatic-microbial pre-purified effluent is fed to a standard size secondary settling basin to segregate the resultant sludge and the other precipitable substances, and to an activation stage for further treatment, where the biological degradation of the BSB, residual burden is effected to the desired limit value.
Example 5100 m3/hour effluent with a BSBs value of 400 mug/1 and at a temperature of 180C are aerated in an activation tank for 30 minutes with 100 m3/h activated sludge. During this time, the effluent constituents are adsorbed by the sludge flocks. In a secondary settling tank, the activated sludge is segregated. Thereafter, the activated sludge is mixed with 2 g/m3 of a mixture of the di-ammonium- and tri-ammonium salt of nitrile tri-acetic acid for 30 minutes. The activated sludge, with an addition of 4 g/m3 of an enzyme preparation, is piped to an enclosed aeration basin which has a temperature of 32 C. After 60 minutes aeration, the activated sludge is piped back to the activation basin.
Example 6Under sterile conditions in two 5000 litre pre-fermenters, at 24 C, on a variant of the maize/spring water/lactose nutrient medium, and in the presence of 10 g of a mixture of di-ammonium salt and 10 g of the tri-ammonium salt of nitrile tri-acetic acid, batchwise growing, and about 20 hours old submerse cultures of penicillin species are added alternately at intervals of about 10 minutes to a 5000 litre capacity main fermenter in continuous manner, the said fermenter already containing fermentation medium pre-heated to 35 C. The fermentation time in the main fermenter is about 12 hours, i.e. that is the time at which the penicillin formation is at maximum.
For every 100 litres of culture medium supplied from the pre-fermenters, 0.1 g of hydrolytic enzyme, such as fi-gluconase, #i-amylase, protease and lipase, pre-dissolved in 100 fold tap water, is constantly added.
The fermentation medium is circulated constantly by pumping during the non-sterile procedure, and the necessary fermentation temperature is maintained through the application of thermal energy (e.g. steam).
In other words, every 24 hours, 10 000 litres culture medium are fed from the prefermenters to the main fermenter, representing a throughput of 10 000 litres per day. Compared with the normal penicillin fermentation, which generally takes 100 hours, the fermentation time with the two-phase process takes only 36 hours.
The fermentation medium continuously withdrawn from the main fermenter, is piped to the preparation stage.
The predominantly autolysed mycel, and due to the hydrolytic action of the injected enzymes, the scarcely hydrophilic colloid contained in the fermentation medium, poses no difficulties during filtration.
The main fermentation can take place continuously over several days, while the pre-fermenter used only for the multiplication of the mycel, has only to be started-up again about 20 hours before its use, so that for the continuous charging of the 5000 litre main fermenter, three pairs of pre-fermentaters are necessary, bearing in mind the pertinent decomposition and preparation times.
Example 7100 m3 effluent (BSB5=300 mg/1, P=10 mug/1) are aerated in the activated sludge tank 1 for 4 hours. During this time, the biomass, containing acinetobacteria, absorbs about 5%P. In the post-settling tank 2, the purified waste water (BSB5=30 mug/1, P=1 mug/1) is separated. 50 m3/d recycled sludge are recirculated without treatment. 50 m3/d are mixed with 0.01% di-ammonium salt of nitrile triacetic acid in tank 5 for 20 minutes and then fed into reactor 3 while adding 0.02% hydrolytic enzymes (referred to in each case to the organic dry substance). Reactor 3 operates at a temperature of 30 C. After 40 minutes, the sludge is again piped back to the activated sludge tank 1.
Example 8100 m3 effluent (BSBs=200 mg/1, P=1Omg/1) are aerated in an activated sludge tank 1 for 4 hours under medium sludge burdening with about 100% recycled sludge. During this time, the circulating and the newly formed biomass absorbs phosphate up to a content of 1.3% P. In the post-settling tank 2, the purified waste water (BSB5=30 mg/1,P=1 mug/1) is separated, and 86.2 m3/d of the recycled sludge are piped in circulation without treatment. Every 30 minutes, 13.8 m3/d are mixed with 0.01% di-ammonium salt of nitrile tri-acetic acid in tank 5, and fed into the Stripp reactor 3 under addition of 0.02% hydrolytic enzymes.
This reactor 3 operates at a temperature of 35 C. After 1 hour, the sludge is fed into the "flotator" 4, where about 7.5 m3/d sludge water with 120 mg/1 P is segregated. 6.3 m3/d are recycled to the activated sludge tank 1.