THEREOF

Technical Field of the Invention

The present invention relates to formation of mixed bacterial culture products in powder form that has a high biodegradable efficiency and is biologically environment friendly, in the presence of an appropriate substrate according to hydrocarbon types for the purpose of being used in the applications of biological treatments of various petrochemical and petroleum refinery plants.

In particular, the present invention relates to production of mixed bacterial cultures in powder form with high efficiency and performance adapted to be used in treating pure terephthalic Acid (PTA) wastewater with a hard-to-biodegrade xenobiotic characteristic by means of reproduction in the presence of appropriate substrates in reactor systems.

State of the Art

There appeared a need for a new generation of applications to avoid pollutants upon the emergence of environmental pollutants. Thus, Environmental Biotechnology is developed as a multi disciplinary science. Environmental Biotechnology practices employ microorganisms with microbial biotechnological potential in eliminating the contaminations resulting from the industrial, agricultural, mining, pharmaceutical etc. originated pollutants . Thus, development is ensured for improving soil, purifying gas, treating surface and underground water and industrial wastewater . The microorganisms are able to degrade the hydrocarbons which are both naturally formed and are included in the structure of the environmental pollutants. It has been found through the studies conducted so far that microorganisms are capable of degrading phenol, benzoate, eicosane, 2 , 4-dichlorophenoxyacetic acid (2,4-D), polyaromatic hydrocarbons (PAH), xenobiotic hydrocarbons and various pollutants in crude oil under proper conditions .

The patent document numbered "US2017313948" has been examined within the result of the preliminary search conducted in the state of the art. The abstract of the invention subject to the application discloses "a process of biodegradation of hydrocarbons using a plant growth promoting rhizobacteria (PGPR) consortium mixed with petroleum wastes, wherein the PGPR consortium is created by inoculating a nutrient broth with pure culture of isolated bacterial strains of Bacillus pumilus, Bacillus subtilis, Pseudomonas putida and Exiguobacterium at Optical Density 1 at 660 nm and bacterial density of 106 cells/ml, and incubating the inoculated nutrient broth". However, method used in said document only allows for working with bacteria consortium of liquid phase under optimized conditions and it is ensured to treat petroleum wastes comprising hydrocarbons, particularly nCll-nC300 types. Therefore, it constitutes some main disadvantages such as not being able to provide long-term storage due to the general hydrocarbon bio degradability and to working in liquid form only.

After preliminary search about the state of the art, the patent document numbered "US4850745" has been examined. The Abstract of the invention subject to the application discloses "a system for treating soil contaminated by petroleum hydrocarbons which comprises applying a suitable bacterial culture capable of degrading petroleum hydrocarbons in a dry form to the bottom of an excavated cavity, applying gravel to a suitable depth over said dry cultures, providing a system of distribution piping capable of supplying nutrients directly to said cultures, and also an air flow through the area containing said cultures, and placing the appropriate tank in place within said cavity and covering with gravel and dirt". Even though disclosed system provides an appropriate medium able to work with dried bacteria cultures on petroleum hydrocarbons, the distribution pipe system formed for the optimum medium condition in which those bacterial cultures may work and use of a tank comprising graven and sand supplements exhibit a limiting effect on both physical conditions and hydrocarbon volume.

The patent document numbered "US5902734" has been examined as a result of the preliminary search conducted in the state of the art. The invention subject to the application discloses a preparation of an isolated mixed bacterial culture, preferably BC-1, ATCC No. 202057, which degrades ethers, especially branched alkyl ethers including MTBE, under aerobic conditions.

Considering the state of the art through inventions listed above, it is seen that activation of products or methods (efficiency, productivity) for the purpose of treating wastewater have a direct relation with medium conditions, the current form (phase) of the effecting bacterial culture and the structure of the effected hydrocarbon component. Accordingly, due to the aforementioned disadvantages and insufficiency of the existing solutions regarding the high-efficiency biological treatment of PTA wastewater, the need for a development in the related technical field has arisen.

Object of the Invention

The most important object of the present invention is to produce a bacterial culture in powder form with high biodegradation efficiency and is adapted for PTA waste water treatment, and to reproduce and render it into a stabilized bacterial culture with prolonged shelf life.

Another object of the present invention is to accelerate the efficacy of said bacterial cultures by means of their easy adaptation to the system on which they affect.

Another object of the present invention is to reduce costs for the process by means of its high biological biodegradation efficacy and system adaptation. (Production is made in a cost- efficient manner.)

The structural and characteristic aspects as well as all the advantages of the present invention will be understood more clearly by means of following figures and the detailed description written by making references thereto. Therefore, assessment should be done by taking these figures and detailed description into consideration.

Description of the Figures

FIGURE 1 illustrates a diagram exhibiting changes of living microorganism concentration according to months relating to shelf life studies.

Description of the Invention

In particular, the present invention relates to production of mixed bacterial cultures in the powder form with high efficiency and performance adapted to be used in treating purified Terephthalic Acid (PTA) wastewater with a hard-to-biodegrade xenobiotic characteristic by means of multiplication in the presence of a general medium in reactor systems.

The inventive stabilized bacterial culture with a high biodegradation efficacy and prolonged shelf life in powder form is comprised of vegetable material, bacterial mixture isolated from active sludge, growth medium components and carrying material as basic components.

The special bacterial mixture isolated from the active sludge comprises the families Pseudomonadaceae, Flavobacteriaceae, Micrococcaceae, Comamonadaceae, Aeromonadaceae, Moraxellaceae . These families are the most common organism groups found in said active sludge, and they are obtained by being isolated from the present wastewater treatment system which is their natural habitats. Therefore, adaptation of microorganisms to the system in the biodegradation of hydrocarbons by means of oxidation is very easy and quick.

General mediums are sufficient and rich in necessary nutrients for microorganisms, and do not increase the development of a certain microorganism group specifically and ensure to develop a plenty of microorganisms. Medium components protein sources are amino acids, polysaccharides, enzymes, vitamins and minerals .

Carrying material facilitates reproducing, applying and using bacteria by solving, diluting, dispersing and modifying physically the medium without altering functions of the bacteria and without causing any interactions to each other. Commonly used carrying materials in the literature are rice silica, fibrous vegetable materials as well es carriers such as diatom, kaolin, paraffine, skim milk, silica, ground sawdust and cryoprotectants . Said fibrous vegetable material content basically comprises some species of cellulose, lignin fibers and the families Fabaceae and Asteraceae . Cellulose is a polymer that is comprised of glucose in the b form and is the most common around the world. 40% of cell walls of plants is comprised of cellulose. It has not only a structural function but also constitutes an important food source for bacteria and fungi. Yet, lignin is a polymer found in vascular plants and some algae as a structural component. Lignin is particularly an important component during the secondary thickening and formation of cell walls in vascular plants. Cellulose and lignin fibers are important as they are held by bacteria and protect their liveliness as well as they are not only natural but also easily degradable .

Preservative material is used for the purpose of protecting bacteria from negative effects resulting from freezing. In the freeze-drying method, the preservative material selection constitutes an importance in protecting, storing and reviving bacteria throughout the lyophilization process. Various polyols, polysaccharides, disaccharides, monosaccharides, amino acids, protein hydrolysate, proteins, minerals, organic acid salts and vitamin complex mediums are used as preservative materials.

The most common preservative materials are skim milk, sucrose, trehalose, sodium glutamate, polyvinyl alcohol, maltodextrin, lactose, maltose, fructose, sorbitol, cellulobios, dextran, glycerol .

Two different methods are employed so as to obtain the product.

1. Carrier-free product in the powder form

Components and amount of the multiplied bacteria isolates are given on Table 1.

Table 1: Table showing the components and amounts of the carrier-free multiplied bacteria isolates.

The main element of the present invention is components given on Table above relating to the product content, wherein stabilized bacterial culture in the powder form, being the final product, is obtained through the production method of which process steps are provided below:

• The families Pseudomonadaceae, Flavobacteriaceae,

Micrococcaceae, Comamonadaceae, Aeromonadaceae,

Moraxellaceae are obtained by isolating the bacteria mixture from the active sludge such that it is in a range of 10-50%.

• In the ambient temperature of 27°C-30 °C, bacteria growth medium component that feeds said families are added into the isolated bacteria mixture in the rate of 10-50%.

• The preservative material in the rate of 10-50% is added into the medium after the predetermined growth ( 10— 10 ¹⁰ kob/ml in the aqueous culture medium) .

• Afterwards, it is lyophilized at the temperature of -50 °C and under the vacuum of 0.1 mBar, thereby obtaining the powder product.

The product obtained through said process steps are studied within the scope of its shelf life at the temperature of +4°C. Studies pertaining to its shelf life is started such that microorganism initial concentration is at least of 10 ¹⁰ kob/ml, and it is determined that its number is of 10 ⁴ -10 ⁸ kob/ml at the end of 24 months. Accordingly, a shelf life of 24 months was determined at the temperature of +4°C being the storage condition .

2. Carrier-containing product in the powder form

Components and amount of the multiplied bacteria isolates are given on Table 2.

Table 2: Table showing the components and amounts of the

carrier-containing multiplied bacteria isolates

The main element of the invention is that the components given on Table above relating to the product content, wherein the stabilized bacterial culture in the powder form, being the final product, is obtained through the production method below: Obtaining bacterial culture in the powder form comprises the process steps of;

• The families Pseudomonadaceae, Flavobacteriaceae, Micrococcaceae, Comamonadaceae, Aeromonadaceae,

Moraxellaceae are obtained by isolating the bacteria mixture from the active sludge such that it is in a range of 10-50%. • In the ambient temperature of 27°C-30 °C, bacteria growth medium component that feeds said families are added into the isolated bacteria mixture in the rate of 10-50%.

• The preservative material in the rate of 10-50% and the carrying material in the rate of 10-50% are added into the medium after the predetermined growth ( 10— 10 ¹⁰ kob/ml in the aqueous culture medium) .

• Afterwards, it is lyophilized at the temperature of -50 °C and under the vacuum of 0.1 mBar, thereby obtaining the powder product.

The product obtained through said process steps are studied within the scope of its shelf life at the temperature of +4°C. Studies pertaining to its shelf life is started such that microorganism initial concentration is at least of 10 ¹⁰ kob/ml, and it is determined that its number is of 10 ⁸ kob/ml at the end of 24 months. Accordingly, it is determined a shelf life of 24 months at the temperature of +4°C being the storage condition. Diagram of the changes pertaining to the numbers of living microorganism according to months in the shelf life studies are shown on Figure 1.

It is ensured to determine lyophilization and different storage conditions such that degradability and liveliness activities of bacterial cultures multiplied by performing pre-tests on the powder product with a high biodegradation efficiency for the inventive PTA wastewater is of the most appropriate. It is evaluated the degradability activity of the powder products comprising the produced microorganisms pertaining to chemicals forming PTA wastewater such as terephthalic acid (TFA) , 4- carboxybenzaldehid (4-CBA) and paratoluic acid (p-tol) at the end of the shelf life. It is aimed to use the inventive bacterial culture in the powder form in biological treatment plans, petroleum industry and pure terephthalic acid (PTA) wastewater treatment.

Preliminary tests were performed in the study and the most proper rates of contents were determined. Therefore, these percentage rates are important in terms of the bacterial liveliness concentration of the final product and any changes in the rates may result in unexpected decreases not only in the final product but also in environmental system, when considering the bacterial liveliness. Accordingly, there will be no improvement in the performance of the treatment process that is the absolute aim.