FR2482130A1 - Inoculum for septic tanks and purificn. digesters - prepd. by drying bacterial cultures with molecular sieve, then adding enzyme and dispersant - Google Patents

Abstract

Prodn. of an inoculum (A) comprises dehydrating cultures of appropriate microorganisms by adding a hydrophilic molecular sieve, so as to temporarily inactivated them, then adding enzymes specific for lysis of long-chain organic material, anhydrous excipients and a powdered nonionic dispersant (I). Pref. dehydration is with a zeolite used at 4-8 wt. times on the cultures and (I) is esp. stearyl alcohol condensed with about 50 units of ethylene oxide. Specified enzymes are protease, amylase, lipase and cellulose, and the pref. microorganisms are Enterobacter cloacae (EC); Pseudomonas fluorescens (PF); Serratia marcescens (Sm); Bacillus cereus (BC) and B.Subtilis (BS). (A) themselves are also claimed. (A) are used for re-equilibration of septic tanks and effluent purificn. digesters. They have high specific activity, good storage properties, and unlike freeze-dried formulations, can be mass prod. inexpensively. The addn. of (I) ensures rapid dispersal.

Description

 The invention relates to a process for the preparation of an inoculum for the biological rebalancing of septic tanks, effluent treatment digesters and the like, according to which specific enzymes are added to dehydrated cultures of selected microorganisms. lysis of long chain organic matter, and excipient charges. The invention also relates to inocula thus prepared.

 The biological degradation of organic matter in effluents, as it occurs in septic tanks, digesters for purifying effluents, is carried out by the action of micro-organisms, bacteria in particular, with methane as essential ultimate terms, carbon dioxide and water, accompanied by mineralized sludge. Basically, this degradation takes place in two stages, a lysis of long-chain organic materials under the action of exoenzymes or coenzymes secreted by microorganisms in the medium which is called substrate, this lysis producing lysates formed mainly of sugars, then consumption of lysates by microorganisms and rejection of ultimate degradation products. Of course effluents almost always contain a certain proportion of organic compounds directly consumable by microorganisms. The biological activity of microorganisms, and in particular their multiplicative reproduction, is closely linked to the consumption of nutritive compounds, lysates and directly consumable compounds, and to the concentration in the substrate of the ultimate degradation products. Due to the short multiplicative reproductive cycle of microorganisms, and if the contact time of the substrate with microorganisms is long enough, the population of microorganisms, or biomass, adapts to the amount of compounds nutritious present in the substrate; biomass and substrate are practically in biological equilibrium.

 In addition, the microorganisms belong to different species which preferentially consume certain organic compounds. Certain species reject waste which is consumable by others, giving rise to symbiotic associations. Biological balance is established within symbiotic associations, and through competition from associations.

However, it sometimes happens that this biological equilibrium is destroyed, either that the flow of supply of organic matter, or flow of influent, undergoes too brutal variations, or that the contribution of matter the degradation of which presupposes prior lysis is excessive in front of the quantity of exo-enzymes that the biomass is able to secrete (starches, proteins, fats-cellulose), that is to say that the influential ones contain toxic products for the biomass (detergents and chlorinated products, such as bleach ) or reproductive inhibitors (antibiotics). 51 then occurs either an accumulation of non-degraded materials, or imbalances in symbiotic associations with decline of species downstream of the symbiotic chains, whose actions are more or less cumulative. Accumulations of non-degraded materials tend to block the oxygenation of the substrate, and aerobic microorganisms wither away. For example, compounds containing nitrogen, sulfur and phosphorus undergo reduced degradations, with the formation of amines, sulfides and phosphides.

 To remedy these imbalances, which result in faulty functioning of the pits, it has been necessary, for a long time, to proceed with cleaning, in order to eliminate the compounds which are factors of imbalance. It has also been proposed to re-seed the pits with inocula, composed of cultures of selected microorganisms, to correspond substantially to the bacterial flora present in the pits in good functional equilibrium. Like the stability of cultures, where the microorganisms are in activity, is only possible if the culture substrate is in constant renewal, the inoculums must contain micro-organisms placed in a state of lethargy, where the exchanges of nutrition and excretion, as well as reproduction, are suspended, without the micro-organisms being killed so far, in order to resume their activity in contact with a favorable environment. Most microorganisms are in lethargy when their osmotic exchanges with the ambient substrate are blocked, in particular by freezing or dehydration. Conventionally, in order to obtain stable inocula, the cultures of microorganisms are subjected to freeze-drying. However, freeze-drying employs complex material of relatively low processing capacity, and consequently constitutes an operational stage the cost has an annoying effect on the overall cost of a product intended for mass distribution.

 In addition, the conservation of lyophilized cultures supposes that they are kept safe from air humidity; as a result, these lyophilized cultures are generally enclosed in doses in capsules, which will be subsequently absorbed into the substrate, like ingestible drug products.

It is known to add to the cultures of microorganisms dehydrated by lyophilization of the enzymes specific for the lysis of the main long-chain organic constituents, the excess of which causes disruption of biological balance in the septic tanks. These long-chained constituents are mainly fats, protein materials, starches and celluloses, so the enzymes added are lipase, protease, amylase and cellulaseO
The action of these added enzymes accelerates the degradation of long-chain organic materials1 so as to enrich the substrate with lysates directly attackable by microorganisms, which can thus rapidly profile and then exert a sufficient quantity of co-enzymes to restore the biological balance.

 In addition, the active components of inoculums, dehydrated cultures of microorganisms and enzymes, must be provided in quantities which represent very low volumes and weights, the dosage of which is difficult. Also, excipients are added in the form of powdery fillers .

 Furthermore, the rapid restoration of biological balance implies rapid dissemination of the product in the volume of the substrate. In this regard, the presentation of the product in capsules is unfavorable, the dissemination can only occur after absorption of the envelope, which n1 is obtained practically only after the agitation of the substrate, following the arrival of the flow of water driving the capsule, has stopped. In addition, dispersion is frequently slowed down by the presence of non-degraded materials (fats, starches).

The presentation of the product in solution, favorable to dispersion, is disadvantageous in terms of conservation since the populations of microorganisms, which are made active by the presence of water without finding a suitable nutritive composition there, regress quickly.

The subject of the invention is a process for the preparation of inocula for the biological rebalancing of septic tanks and digesters, which combine great specific activity with good preservation.

A subject of the invention is also a process for the preparation of inexpensive docs in mass production.

 For these effects, the invention provides a process for the preparation of an inoculum intended for the biological rebalancing of septic tanks and digesters for purifying effluents, according to which cultures of selected microorganisms are put into lethargy by dehydration, specific enzymes for lysis of long-chain organic materials, and optionally fillers of excipients, characterized in that said cultures are dehydrated by the addition of a sufficient quantity of a hydrophilic molecular sieve, and in that, in addition the charges of anhydrous excipients, a powdery nonionic dispersant is added.

The applicant has discovered that the dehydration of cultures of microorganisms by contact with a hydrophilic molecular sieve, that is to say adsorbent for polar molecules with a diameter of the order of magnitude of the water molecules, allows an effective lethargy of the selected microorganisms, by a process easy to implement on an industrial scale and rapid in itself, without appreciable destruction of these microorganisms. Furthermore, the addition to the charges of a powdered nonionic dispersant slow allows, in use, to ensure rapid dispersion of the inoculum in the substrate to be treated, without the conservation before use being affected by the presence of water provided by the dispersant, as would happen with a dispersant usual in aqueous solution.

 Preferably, the molecular sieve is a zeolite which is added to the cultures in a weight ratio of 4 to 8, which corresponds substantially to the water adsorption capacity of the zeolites.

The origin strains of the preferred cultures are chosen from species normally present in sepJGioues ditches or digesters, namely Enterobacter Gloacas, Pseudomonas Fluorescens, Serratia Marcescenss
Bacillus Cereus and Bacillus Subtilis, which will be frequently abbreviated below by EoCoD PaFo SM respectively,
BC, and BS

Preferably also the enzymes will be chosen9 as a function of the difficultly degradable organic materials present, among the protease, the lipase, the amylase and the cellulose.

 In a preferred method, a mixture of cultures from distinct strains will be composed immediately before use. The molecular sieve will be dispersed by stirring in the mixture, and the enzymes, the nonionic dispersant and finally the charges of excipients will be successively added.

 The cultures are prepared, in preferred arrangement, by seeding with the corresponding strain of a substrate comprising, per liter, 4 g of glucose, 3 g of yeast extract, 3 g of beef extract, 5 g of sodium chloride and 2 g of starch, dissolved in slightly mineralized spring water, then incubation at 370C until suitable development of the culture, controlled by population count. This gives well reproducible cultures.

 The characteristics and advantages of the invention reso will emerge from the description which follows, together with examples.

Initially, it was necessary to review the problem of the biological rebalancing of septic tanks and effluent digesters in all its aspects, that is to say the operating conditions in equilibrium, the manifestations and causes of imbalances, and the inadequacies of solutions provided in the state of the art. These aspects have been mentioned above.

It then appeared that the improvements had to relate essentially to two points: the seeding of septic tanks and digesters with cultures of specific microorganisms in a state of effective and contral activity, and the establishment of conditions favorable to multiplication microorganisms in the moments after sowing.

On the first point, it was clear that sowing had to be carried out with selected cultures, at a stage preceding degeneration. The known technique consisting in fixing the growth state of the culture by freezing, followed by lyophilization to allow the lethargy of the culture to be maintained and its conservation in the fixed growth state, a technique which has been developed for biomedical and pharmaceutical applications of microorganisms, appeared to be not very compatible with industrial production, as much by its cost price as by the difficulties of handling and dosing of lyophilized cultures due to the low inherent volume of microorganisms. Furthermore, freeze-drying is very well suited to the specific requirements of pharmaceutical and biomedical applications, a privileged field for the use of micro-organisms with metered effects, the state of the art did not offer a replacement process for freeze-drying for putting in lethargy of microorganisms.

It was known to separate the constituents of a solution according to the size of their molecules by means of porous adsorbent bodies called molecular sieves, the diameter and the surface chemical functions of the pores allow a selective adsorption of molecules, in particular polar, whose diameter is less than the diameter of the pores.

The molecular sieves presenting a selectivity for the adsorption of polar molecules of size comparable to that of water molecules will be called hydrophilic. A particular class of molecular sieves is formed by zeolites, or hydrated silicates
The Applicant has discovered that the water adsorption properties of molecular sieves can be used with success in the dehydration of microorganisms, that the microorganisms thus put into lethargy resume their normal activity in contact with a suitable amtritive substrate. , and that the presence of the molecular sieve then had no undesirable side effects ##, and on the contrary facilitated the metered dispersion of the microorganisms by acting as a diluent. diluent As molecular sieve which is well suited to the application considered, there may be mentioned the artificial zeolite marketed by CECA under the name of "Siliporite NE 10'l.

 Furthermore, it has been observed that dehydration takes place under the best conditions when the water adsorption capacity of the quantity of molecular sieve corresponds substantially to the quantity of water in the culture used. that, in culture, the proper volume of microorganisms is negligible compared to the volume of water, and that the adsorption capacity of molecular taxa is within a range of weight ratio molecular sieve / water from 4 to 8 (substantially 5 for the above-mentioned zeolite), the typical dehydration process comprises the addition, in a high speed mixer, to a determined mass of cultures, of 4 to 8 times this mass of molecular sieve and stirring until complete apparent dryness.

 The choice of strains of microorganisms was guided by the following considerations. The cultivated species must correspond to the predominant species in septic tanks or digesters, and the strains must be readily available and sufficiently stable for the cultures to be reproducible; finally, it is desirable to choose strains belonging to non-pathogenic subspecies.

The strains selected by the depositor are, in the classification of the Institut Pasteur - Paris (EC) Enterobacter Cloacae 60.22 (BC) Bacillus Cereus A.30 CIP (PF) Pseudomonas Fluorescens 56.90 (SM) Serratia Marcescents 53.101 (BS) Bacillus Subtilis 53.159
It should be noted that if certain Bacillus subspecies
Subtilis are considered to be pathogenic, there are a number of strains corresponding to non-pathogenic subspecies, and the selected strain mentioned above is particularly safe in this regard.

EXAMPLE 1
Culture process.

The process includes preparing the culture medium, seeding and incubation, and controlling population density. Each strain must be grown separately. The composition of the culture medium and the incubation conditions are, however, practically the same.

A) Culture medium.

4 about 3/4 of a liter of lightly mineralized spring water (Evian water type) add 4 g of glucose, 3 g of yeast extract (THANK YOU), 3 g of beef meat extract ( THANKS), 5 g of sodium chloride and 2 g of starch after dissolution of the additions, make up to one liter with the same spring water.

B) Seeding.

 A sterile container containing a known volume of culture medium is placed in an incubator at a temperature set at 370 + 0.50C. When the culture medium has reached the temperature of the incubator, a sample of the desired strain is introduced into the container. This was transplanted, from the lyophilisate received, on agar under conventional conditions, in accordance with the general instructions of the organism supplying the strains.

C) Incubation.

The temperature of the incubator being maintained at 370 + 0, 50C. The state of the culture is periodically checked by count, as will be specified in the following paragraph
After a latency period of approximately 2 hours, during which the population does not undergo appreciable growth, there is a period of ex ponential growth, approximately 8 hours, then a gradual slowing down of the population. rate of growth. After an incubation of about 36 hours, the culture will have undergone its full development, then will begin a regression phase. We therefore arrive at the culture a little before its full development O D) Control of population density.

The population densities corresponding to the optimal time of shutdown have been determined experimentally. These, expressed in silliards of individuals per gram of culture medium, were fixed at
6 for EC Enterobacter Cloacale
5 for PF Pseudomonas Fluorescens
5 for S * M. Serratia Marcescents
4.7 for BC Bacillus Cereus
2 for B.8. Bacillus Subtilis.

 To control the population density, we first operate by successive dilutions of a culture sample, to arrive at an estimated density of a few hundred individuals per milliliter. A count is then made by the "Nillipore" method, with absorption of 1 ml of culture diluted in a filter, and counting of the colonies which develop from each individual on the surface of the filter.

The cultures are stopped by the following process
The extemporaneous mixing of determined quantities of selected cultures is carried out, according to the intended application9 to correspond substantially to the population distributions in a digester in biological equilibrium 9 and this ge mixing is placed in a high speed mixer, Nauta type, and the adding a weight of Siliporite NX 10 equal to 5 times the weight of the mixture. Leave to run until apparent dryness.

EXAMPLE 2 Dehydrated cultures for domestic septic tank inoculum.

 The culture mixture comprises 4.4 g of EC culture, 1.4 g of BC, 7.4 g of BS, 3.4 g of PF and 3.4 g of SM. 100 g of Siliporite NE 10 are added. .

EXAMPLE 3
Dehydrated cultures for digester inoculum.

The mixture comprises 15.7 g of EC culture, 12.15 g of FP and 12.15 g of SM. Two hundred grams of
Siliporite NX 10.

At the end of the dehydration of the cultures, specific enzymes for the lysis of organic materials with long chains are introduced, chosen to correspond in specificities and quantities to the organic materials present in influent pits or digesters, and the accumulation of which is the cause of biological imbalance. These enzymes are essentially protease (500 ST), for protein lysis, amylase (THC 250), for starch hydrolysis, lipase (80,000 ST), for fat hydrolysis, and cellulase ( 50,000 SU) for the hydrolysis of cellulose from paper and food residues.

After the introduction of the enzymes, a nonionic dispersant is added, consisting of a condensate of stearoyl alcohol with a polyethylene oxide containing approximately 50 units, that is to say resulting from the polymerization of approximately 50 molecules of ethylene oxide. This dispersant is marketed by Witco Chemical under the name of Cetalox AU.

 After that add loads of excipients, including baking soda, dabbing, and sodium chloride. The preferred inoculum compositions will be specified below. On the whole, these charges will have the role of ensuring, during the dispersion in the substrate to be treated, that the composition of the substrate in contact with the micro-organisms is favorable to their resumption of activity, and possibly to cooperate with the dispersant to ensure rapid and effective dispersion. In addition, these charges make it possible to easily define doses of cultures and enzymes.

EXAMPLE 4
Inoculum composition for domestic septic tanks.

To the dehydrated culture of Example 1 (120 g) is added 1.200 kg of protease, 0.120 kg of amylase, 0.004 kg of lipase and 0.060 kg of eellulase, then 2 kg of FGetalox AI, 5 kg of sodium bicarbonate , 10 kg of tale, and 81.5 kg of an equal parts mixture of micronized powder mica and iodine-free sodium chloride. 100 kg of powdered inoculum are thus obtained.

 For normal maintenance of a conventional septic tank, corresponding to the use by 3 people, approximately 25 g of this inoculum will be deposited every week, and it will be drawn into the tank by a flush.

EXAMPLE 5
Inoculum composition for effluent digesters.

To the dehydrated culture of Example 2 (240 g) 0.090 kg of lipase (80,000 ST) is added, then 6 kg of i'Cetalos
AT ", and after 30 minutes, 0.500 kg of sodium bicarbonate, 14 kg of disodium phosphate (anhydrous), 14 kg of sodium phosphate, and 15.17 kg of sodium chloride, to obtain 50 kg of inoculum.

 It will be noted that this inoculum is intended for cation supports where the fats constitute the major engorgement agent. In addition, phosphates promote the reaeltiva tion of microorganisms.

This inoculum finds its use in municipal settling tanks, the maintenance of drains, grease traps, garbage disposers, sinks, sumps, the maintenance of fish orchards (treatment of pig manure) in slaughterhouses. .

 Excluding shock treatments, for the rehabilitation of very biologically unbalanced installations, where relatively high doses can be used for a massive action of lipase, we will count for normal maintenance about lg per m3 every 20 days.

It will be noted that the charges of excipients have a role only during the inoculation operations of the substrate, to facilitate the determination of the doses, and possibly locally correct the composition of the substrate. As a result, the active product, consisting of the cultures dehydrated by the molecular sieve, the enzymes and the pulverulent dispersant, can be prepared and stored without loads of excipients. During a shock treatment requiring high doses of active product, the presence of loads of excipients can prove to be at least useless, if not harmful, since the dosing on the active product no longer presents difficulties while the fillers could form sludge in the substrate, or provide excessive concentrations of soluble salt. We would then prefer to use the pure active product, as it appears in Examples 4 and 5 before adding sodium bicarbonate. Furthermore, since the addition of loads of excipients does not present any particular difficulties, it will be possible, to avoid long-distance transport of heavy products, to send the pure active product to a distant country where the addition of loads of excipients before marketing.

 For the control of the effectiveness of the inocula, the depositor has defined a standard substrate, in order to determine the speed of digestion of the various components and the evolution of the populations of microorganisms.

This substrate has the following composition, by weight
Water 93.4%
Lard 0.25%
Soap 0.10 *
Wheat starch 3.45%
Cellulose 0.10%
Gelatin 2.70 *
The mixture is stirred vigorously, then left to stand for 24 hours. The viscosity, determined with a DRAG viscometer equipped with a mobile A, and taken at three speeds of rotation V1, V2 and V3 is established at
V1 13.549 Pa.s
V2 6.116 Pa.s
V3 2,387 Pitch
Note the decrease in viscosity with the increase in rotation speed of the measuring mobile.

 By adding to the substrate, maintained at an average temperature of about 150C 0.1% by weight of the inoculuni according to Example 3, the viscosity after 24 hours drops to 5.4 10-3 Pa.s. It is recalled that the viscosity of pure water at 20 C is substantially 10-3 Pa.s.

 Of course, the invention is not limited to the examples described, but embraces all the variants thereof. In particular, the quantities of enzymes added to the cultures can be adjusted, for particular hare applications, in proportion to the barley contents of long effluents, the charges of excipients can be adapted to compensate for deficiencies of these effluents in nitrogen or phosphorus.

Claims (12)

 1. Process for the preparation of an inoculum intended for the biological rebalancing of septic tanks and digesters for purifying effluents, according to which are added to cultures of selected microorganisms, put into lethargy by dehydration, specific enzymes for lysis of organic materials with long chains, and optionally fillers of excipients, characterized in that said cultures are dehydrated by adding a sufficient quantity of a hydrophilic molecular sieve, and in that, in addition to the fillers of excipients, anhydrous , a powdery nonionic dispersant is added. 2. Method according to claim 1, characterized in that said molecular sieve is a zeolite. 3. Method according to claim 2, characterized in that said zeolite is added to the cultures in a weight ratio of 4 to 8. 4. Method according to claim 1, characterized in that the nonionic dispersant is a condensate of stearoyl alcohol with a polyethylene oxide with approximately 50 units. 5. Method according to claim 1, characterized in that said cultures come from strains of microorganisms chosen from the group comprising Enterobacter Cloacae (E.C.), Pseudomonas Fluorescens (P.F.), Serratia Marcescens (S.fl.), Bacillus Cereus (B.C.) and Bacillus Subtilis (B.8.).  6. Method according to claim 1, characterized in that said enzymes are chosen from the group comprising protease, amylase, lipase and cellulase.  7. Method according to claim 1, characterized in that a mixture of cultures of distinct microorganism strains is composed immediately, the molecular sieve is dispersed by stirring in the culture mixture, then the enzymes, the powdery non-ionic dispersant and finally the excipient charges.  8. Process according to claim 7, including the preparation of said cultures, characterized in that a substrate comprising, per liter of microorganisms, is seeded comprising, per liter, 4 g of glucose, 3 g of yeast extract , 3 g of beef extract, 5 g of sodium chloride and 2 g of starch, dissolved in the required amount of lightly mineralized spring water, and the seeded substrate is incubated at a temperature of 370 s O, 50C for 12 to 36 hours.  9. Method according to claim 8, characterized in that the incubation is terminated when the population density of microorganisms in the substrate reaches a chosen value. 10. Method according to joint claims 5 and 8, characterized in that the chosen value of population density, expressed in billions of individuals per gram of substrate, is approximately 6 for EC, 5 for PF and SM, 4, 7 for BC and 2 for BS, 11. inoculum for domestic septic tanks prepared by the method according to claim 10, characterized in that it comprises 20 g of mixture of cultures composed of 4.4 g of EC, 1.4 g of BG, 7.4 g of BS , 3.4 g of PP and 3.4 g of SM, 100 g of zeolite molecular sieve, 1.2 kg of protease, 0.12 kg of amylase, 0.004 kg of lipase and 0.060 kg of cellulase, 2 kg of stearoyl dialcohol condensate with a polyoxyethylene with about 50 units, 5 kg of sodium bicarbonate, 10 kg of talc and 8195 kg of a mixture of equal parts of micronized powder mica and iodine-free sodium chloride.  12. Inoculum for the maintenance of effluent treatment digesters, prepared by the method according to claim 10, characterized in that it comprises 40 g of mixture of cultures composed of 15.7 g of E0C., 12.15 g of FP and 12.15 g of SM, 200 g of zeolite molecular sieve, 90 g of lipase, 6 kg of stearoyl alcohol condensate with polyoxyethylene with about 50 units, 0.5 kg of sodium bicarbonate, 14 kg disodium phosphate, 14 kg monosodium phosphate and 15.17 kg iodine-free sodium chloride.