Microbial-based biological surface-active agent

Abstract

The invention relates to a microbial-based biosurfactant (biological surface-active agent) and the production method thereof. This said natural, biological, environmentally friendly and biodegradable biosurfactant obtained from Pseudomanas sp., which is the subject of the invention, reduces chemical and water usage during leather production.

Description

[0001] MICROBIAL-BASED BIOLOGICAL SURFACE-ACTIVE AGENT

[0002] Technical Field of the Invention

[0003] The invention relates to a microbial-based biosurfactant (biological surface-active agent) and the production method thereof. This said environmentally friendly and biodegradable biosurfactant obtained from Pseudomanas sp., which is the subject of the invention, reduces chemical and water usage during the leather production.

[0004] State of the Art

[0005] The main objective of the leather industry, which plays an important role in today's global economy, is to turn raw hides and skins into a physically and chemically stable material by subjecting them to sequential chemical and mechanical processes, to produce leather goods that meet the consumer demands. Raw hides and skins, which are by-products of the meat and meat products industry, are used as raw materials in the leather industry. In this respect, the leather industry is considered as environmentally friendly, that involves the processing of waste products from meat production [1], However, although this industry is considered to be environmentally friendly, like all industries, it generates waste and about 70% of the environmental pollution in the leather industry comes from the beamhouse processing steps where chemical products are used. In the present art, soaking, and degreasing are important steps in the processing of raw hides and skins. Among these, soaking consists of the raw hides and skins saturating process with water and ensures proper hydration. The soaking process involves the decomposition of proteins in the hide and skin as well as loosening the fibers, which is usually carried out using chemicals such as surfactants, biocides, salts, etc. The degreasing process aims to remove the natural fat from the hide and skin and generally requires the use of a surfactants during the process. Further, raw hide and skin arrives at the leather factory contaminated with various dirt, oil, and other pollutants and these are removed by the use of chemical surfactants during the leather production processes. Therefore, surfactants, known as amphiphilic compounds, that reduce surface and interfacial tension by accumulating between two immiscible phases, are widely used in the state of the art to clean the hide and skin and remove contaminants. Surfactants, generally used with biocides in soaking baths, are used alone in the degreasing process as ethoxylated fatty alcohol-based synthetic chemical surfactants, organic solvents and / or mixtures thereof in aqueous media and constitute the content of the resulting wastewater.

[0006] The non-biodegradable nature, toxicity to aquatic organisms, and flammability characteristics of chemical surfactants, as well as the toxicity of solvents create problems for the environment and human health. Besides, chemical surfactants cause environmental pollution when discharged into water systems and their dissolution and accumulation in water have a negative impact on aquatic life and other organisms. They have harmful effects on aquatic organisms such as fish, surface reptiles and aquatic plants that disrupt the balance of aquatic ecosystems. Also, they accumulate in the bodies of organisms and move up the food chain, eventually causing harm to upper-level consumers, including humans [2], In addition to all these disadvantages of chemical surfactants, biobased amphiphilic molecules (methyl ester sulfonate (MES)) produced from the renewable natural raw materials in the state of the art are widely preferred due to legal regulations, restrictions, and developing public awareness. MES, which is on the market as an environmentally friendly and viable alternative to linear alkylbenzene sulfonate (LAS), the most well-known chemical surface-active agent, is partially chemical-based and therefore incapable of completely eliminating environmental problems.

[0007] Biosurfactants are biologically derived surfactants and produced by microorganisms such as bacteria, yeasts, and fungi. The hydrophilic parts typically consist of polar functional groups such as sugars, amino acids, or carboxylic acid groups, while the hydrophobic part is generally composed of the hydrocarbon chain of p-hydroxy fatty acids. As is known, surfactants are evaluated based on their properties such as surface / interfacial tension reduction and critical micelle concentration (CMC). CMC values, indicating the degree of surface and interfacial tension reduction for various biosurfactants, have been determined in the state of the art. In general, biosurfactants are found more efficient than chemical surfactants, requiring much lower concentrations — typically 10 to 40 times less — to achieve similar surface tension reductions, due to their lower CMC values [3]. Moreover, biosurfactants pose minimal environmental risk, as they are readily biodegradable by bacteria and other microorganisms [4], Studies have demonstrated faster biodegradation rates for biosurfactants such as mannosylerythritol lipid, sophorolipids, and rhamnolipids compared to various synthetic surfactants that are available in the present art [5]. Furthermore, another important property of biosurfactants with industrial and biotechnological uses is that surface activity of biosurfactant is not affected by environmental conditions such as temperature and pH. In addition, biosurfactants can be produced from cheap raw materials that are abundantly available.

[0008] Biosurfactants are classified into 5 major groups based on their chemical composition and microbial origin: glycolipids, lipopeptides, lipoproteins, polymeric biosurfactants, and particulate biosurfactants [6]. These microbial surface-active agents have a broad range of industrial and environmental applications and are derived from diverse microorganisms such as Pseudomonas sp., Burkholderia sp., Mycobacterium sp., Bacillus sp. Among these, species of the genus Pseudomonas sp. particularly Pseudomonas aeruginosa — are well-known producers of rhamnolipids, one of the most extensively studied glycolipid biosurfactants. Rhamnolipids can reduce the surface tension of water to remarkably low values, making them highly suitable for bioremediation of environments contaminated with petroleum or heavy metals [7], In the present literature, P. aeruginosa is reported to produce biosurfactant in stationary phase, and the surface tension values of rhamnolipids produced from Pseudomonas aeruginosa ranges between 26 and 29 mN / m [8,10].

[0009] The limitations and shortcomings of the solutions at present, the use of toxic chemical surfactants in leather production steps such as soaking and degreasing, the non- biodegradable, flammable, and toxic nature of these chemical surfactants and solvents, the resulting high water consumption and wastewater generation, and the associated environmental impacts, particularly the increase in biological oxygen demand (BOD), which promotes algal growth that disrupts aquatic life are the increasingly necessary reasons to develop microbial biosurfactants. Furthermore, the stricter and more restrictive global environmental legislations on the discharge of wastewater worldwide, along with consumer preferences shifting toward bio-based alternatives, have reinforced the demand for sustainable, microbial-derived biosurfactants.

[0010] Summary and Objects of the Invention

[0011] The invention discloses a microbial-based biosurfactant and a production method thereof. The subject of the invention is a natural, biological, environmentally friendly and biodegradable biosurfactant obtained from sustainable sources from Pseudomonas sp. and reduces the use of chemicals and water during leather production.

[0012] The aim of the invention is to convert the soaking and degreasing steps in leather manufacturing into environmentally friendly and sustainable production processes. In the state of the art, chemical surfactants are generally used as aqueous solutions in the soaking and degreasing processing steps in leather production. But the use of the environmentally friendly biosurfactant of the invention does not require additional water usage or dissolution in water. Therefore, the amount of water to be used during the production decreases. Also, its easy-to-rinse nature further lowers the water consumption in the subsequent rinsing steps. Furthermore, since said biosurfactant is used as a soaking agent in the soaking process and as a degreaser in the degreasing process, the need for chemicals to be used during these processes is eliminated.

[0013] The other aim of the invention is to provide an environmentally friendly and biodegradable biosurfactant. Since said biosurfactant is derived from Pseudomonas sp., it is 100% natural and non-toxic, therefore the use of the biosurfactant does not harm the environment or humans.

[0014] Another object of the invention is to reduce waste production occurred due to the leather production. The chemicals used in leather manufacturing processes are also present in the wastewater discharged after production. However, since the biosurfactant of the invention eliminates the need for the use of these chemicals, it also reduces the production of the waste.

[0015] The final object of the invention is to improve the quality of the finished leather. This improvement is achieved through the antimicrobial property of the invention when used as a soaking agent during the soaking step. With this property, it reduces the number of microorganisms in the soaking step, limiting collagen denaturation and positively affecting the quality of the finished leather. Furthermore, using the biosurfactant of the invention in the degreasing step showed a positive impact on color, making the leather appear whiter than the control sample. Detailed Description of the Invention

[0016] The invention relates to a microbial-based biosurfactant and a production method thereof. The invention relates to a natural, biological, environmentally friendly, and biodegradable biosurfactant derived from Pseudomonas sp., which facilitates the conversion of the soaking and degreasing steps in leather production into sustainable and clean technologies, while also reducing the use of chemicals and water throughout the process. Therefore, the amount of waste generated from leather production also appears to decrease.

[0017] The microbial-based biosurfactant of the invention is produced from tryptone, glucose, waste oil, microorganism inoculum culture, distilled water, and yeast extract. In an embodiment of the invention, the biosurfactant of the invention contains 0.5-4% tryptone by volume, 0.5-5% glucose by volume, 0.1 -1 % waste oil by volume, microorganism inoculum culture, distilled water, and 0.1 -1.5% yeast extract by volume. In another embodiment of the invention, the biosurfactant of the invention comprises 2.2% tryptone by volume, 3.2% glucose by volume, 0.6% waste oil by volume, 10% microorganism inoculum culture, 83% distilled water, and 1% yeast extract by volume. Here, said inoculum culture comprises Pseudomonas sp. and said Pseudomonas sp. is Pseudomonas aeruginosa.

[0018] The production method of the microbial-based biosurfactant of the invention comprises the process steps of: i. preparing inoculum culture with tryptone, glucose, waste oil, and yeast extract and sterilizing the prepared culture, ii. adding Pseudomonas sp. to the inoculum culture left to cool and preparing it for bioreactor production in a shaking incubator, iii. preparing bioreactor production media containing tryptone, glucose, waste oil, and yeast extract and sterilizing the bioreactor, iv. after the bioreactor has cooled down, connecting the reactor to its system and operating it to stabilize the oxygen, pH, and temperature probes, v. adding the inoculum, prepared in a shaking incubator, into the bioreactor under aseptic conditions, vi. once bioreactor production is complete, separating the cell-containing biosurfactant liquid from the bioreactor under aseptic conditions and processing it through centrifugation, vii. upon collection of cells at the bottom of the centrifuge tubes, separating the resulting cell-free liquid.

[0019] In an embodiment of the invention, the production method of the microbial-based biosurfactant of the invention comprises the process steps of: i. preparing inoculum culture with 0.5-4% tryptone, 0.5-5% glucose, 0.1 -1% waste oil, and 0.1 -1.5% yeast extract by volume and sterilizing the prepared culture, ii. adding Pseudomonas sp. to the inoculum culture left to cool and preparing it for bioreactor production in a shaking incubator, iii. preparing bioreactor production media containing 0.5-4% tryptone, 0.5- 5% glucose, 0.1 -1 % waste oil, and 0.1 -1 .5% yeast extract by volume and sterilizing the bioreactor, iv. after the bioreactor has cooled down, connecting the reactor to its system and operating it to stabilize the oxygen, pH, and temperature probes, v. adding inoculum culture developed in a shaking incubator to the bioreactor 12-18 hours later under aseptic conditions and a temperature of 30-40°C, pH of 6-8, and air flow rate of 0.5-3 vvm, vi. after completion of bioreactor production, separating the cell-containing biosurfactant liquid from the bioreactor under aseptic conditions and processing it through centrifugation, vii. upon collection of cells at the bottom of the centrifuge tubes, separating the resulting cell-free liquid.

[0020] In another embodiment of the invention, the production method of the microbial-based biosurfactant of the invention comprises the process steps of: i. preparing inoculum culture with 2.2% tryptone, 3.2% glucose, 0.6% waste oil, and 1 % yeast extract by volume and sterilizing the prepared culture in autoclave at 121 °C for 20 minutes, ii. adding 1% by volume of Pseudomonas sp. to the inoculum culture left to cool down at and preparing it for bioreactor production in a shaking incubator at 35°C for 24 hours at 150 rpm, iii. preparing bioreactor production media containing 2.2% tryptone, 3.2% glucose, 0.6% waste oil, and 1% yeast extract by volume and sterilizing the bioreactor in autoclave at 121 °C for 20 minutes, iv. after the bioreactor has cooled down, connecting the reactor to the-system and operating it for 12 hours to stabilize the oxygen, pH, and temperature probes, v. adding inoculum culture, prepared in a shaking incubator, to the bioreactor 12-18 hours later under aseptic conditions and a temperature of 35°C, pH of 7, and air flow rate of 1 vvm, vi. once bioreactor production is complete, separating the cell-containing biosurfactant liquid from the bioreactor under aseptic conditions and processing it through centrifugation at a temperature of 21 °C at 10.000 g for 15 minutes, vii. upon collection of cells at the bottom of the centrifuge tubes, separating the resulting cell-free liquid.

[0021] In the method of the invention, the inoculation made by adding Pseudomonas sp. strain in the process step (ii) is accepted as the starting time and the production completed in the bioreactor is completed between 16-48 hours, optimally at 24 hours. Furthermore, the bacterium used in the invention is Pseudomonas aeruginosa.

[0022] In the method of the invention, the optimized biosurfactant production time occurs in the logarithmic phase of production, not in the stationary phase. While the surface tension values of rhamnolipids produced from Pseudomonas aeruginosa bacteria vary between 26 and 29 mN / m in the present art [8,10], this value of the biosurfactant of the invention was determined as 30 mN / m. Also, the biosurfactant efficiency of the rhamnolipid species of the invention differs from the data reported in the literatures (2 to 10 g / L). A biosurfactant efficiency of 17.4 g / L distinguishes it from the other alternatives. The low concentration of biosurfactants per liter presents a challenge in the product's separation and purification processes. The efficiency of the biosurfactant of the invention overcomes this challenge. In addition, it was determined that biosurfactant has antimicrobial properties when used as a soaking agent in the soaking process. This feature was evaluated against the Proviera brand’s commercially available soaking and degreasing agent, and it was determined that, when used in equal quantities, it reduced the microbial load in the soaking bath by 10%. It was also found that these two products have similar degreasing efficiency. Biosurfactants are also known for their tolerance to extreme pH, temperature, and salinity conditions. The rhamnolipid type biosurfactant produced by the strain of the invention is resistant to different pH values (6, 8 and 10), salt concentrations (2, 4, 6 and 8%) and temperatures (30, 50, 70 and 90°C). It does not show any loss of activity during these change intervals.

[0023] Industrial Applicability of the Invention

[0024] The invention relates to a microbial-based biosurfactant and the production method thereof and is industrially applicable.

[0025] The invention is not limited to the above descriptions and the person skilled in the art can readily present other different embodiments of the invention. These should be considered within the protection scope of the invention claimed by the claims.

[0026] REFERENCES

[0027] [1] Ozgunay, H., Colak, S., & Akyuz, F. (n.d.-b). Characterization of leather industry wastes. Polish Journal of Environmental Studies.

[0028] [2] Arora J;Ranjan A;Chauhan A;Biswas R;Rajput VD;Sushkova S;Mandzhieva S;Minkina T;Jindal T; (n.d.). Surfactant pollution, an emerging threat to ecosystem: Approaches for effective bacterial degradation. Journal of applied microbiology.

[0029] [3] Desai JD, Banat IM. 1997. Microbial production of surfactants and their commercial potential. Microbiology and Molecular Biology Reviews. 61 (1 ):47-64.

[0030] [4] Rahman PKSM, Gakpe E. 2008. Production, Characterisation and Application of Biosurfactants. Biotechnology. 7(2):360-370.

[0031] [5] Kim HS, Jeon JW, Kim SB, Oh HM, Kwon TJ, Yoon BD. 2002. Surface and physicochemical properties of a glycolipid biosurfactant, mannosylerythritol lipid, from Candida antarctica. Biotechnology Letters. 24(19): 1637- 1641.

[0032] [6] Desai JD, Banat IM. 1997. Microbial production of surfactants and their commercial potential. Microbiology and Molecular Biology Reviews. 61 (1 ):47-64.

[0033] [7] Mukherjee, A. K., & Das, K. (1970, January 1 ). Microbial surfactants and their potential applications: An overview. SpringerLink.

[0034] [8] Guerra-Santos, L. H., O. Kappeli, and A. Flechter. 1986. Dependence of Pseudomonas aeruginosa continuous culture biosurfactant production on nutritional and environmental factors. AppL Microbiol. Biotechnol. 24: 443-448

[0035] [9] Robert, M., M. E. Mercade, M. P. Bosch, J. L. Parra, M. J. Espuny, M. A. Manresa, and J. Guinea. 1989. Effect of the carbon source on biosurfactant production by Pseudomonas aeruginosa 44T. Biotechnol. Lett. 11 :871-874.

[0036]

[0010] Sami Ibrahim, Atef Diab, Hesham Abdulla, Bio-cleaning Efficiency of Rhamnolipids Produced from Native Pseudomonas aeruginosa Grown on Agro-industrial By-products for Liquid Detergent Formulation, Applied Biochemistry and Biotechnology (2021 ) 193:2616-2633

Claims

CLAIMS1 . A biosurfactant, characterized by its composition that includes tryptone, glucose, waste oil, microorganism inoculum culture, distilled water, and yeast extract.

2. The biosurfactant according to claim 1 , characterized by the composition of 0.5- 4% tryptone by volume, 0.5-5% glucose by volume, 0.1 -1% waste oil by volume, microorganism inoculum culture, distilled water, and 0.1 -1.5% yeast extract by volume.

3. The biosurfactant according to claim 2, characterized in that it comprises 2.2% tryptone by volume, 3.2% glucose by volume, 0.6% waste oil by volume, 10% microorganism inoculum culture, 83% distilled water, and 1 % yeast extract by volume.

4. The biosurfactant according to any one of claims 1 -3, characterized in that said microorganism inoculum culture comprises Pseudomonas sp.

5. The biosurfactant according to claim 4, characterized in that said Pseudomonas sp. is Pseudomonas aeruginosa.

6. A production method of a biosurfactant, characterized in that it comprises the process steps of: i. preparing inoculum culture with tryptone, glucose, waste oil, and yeast extract and sterilizing the prepared culture, ii. adding Pseudomonas sp. to the inoculum culture left to cool and preparing it for bioreactor production in a shaking incubator, iii. preparing bioreactor production media containing tryptone, glucose, waste oil, and yeast extract and sterilizing the bioreactor, iv. after the bioreactor has cooled down, connecting the reactor to the system and operating it to stabilize the oxygen, pH, and temperature probes, v. adding the inoculum culture, developed in a shaking incubator, into the bioreactor under aseptic conditions,vi. after completion of bioreactor production, separating the cell-containing biosurfactant liquid from the bioreactor under aseptic conditions and subjecting it to centrifugation, vii. upon collection of cells at the bottom of the centrifuge tubes, separating the resulting cell-free liquid.

7. The method according to claim 6, characterized in that it comprises the process steps of: i. preparing inoculum culture with 0.5-4% tryptone, 0.5-5% glucose, 0.1 -1% waste oil, and 0.1 -1.5% yeast extract by volume and sterilizing the prepared culture, ii. adding Pseudomonas sp. to the inoculum culture left to cool and preparing it for bioreactor production in a shaking incubator, iii. preparing bioreactor production media containing 0.5-4% tryptone, 0.5- 5% glucose, 0.1 -1 % waste oil, and 0.1 -1 .5% yeast extract by volume and sterilizing the bioreactor, iv. after the bioreactor has cooled down, connecting the reactor to the-system and operating it to stabilize the oxygen, pH, and temperature probes, v. adding inoculum culture prepared in a shaking incubator to the bioreactor 12-18 hours later under aseptic conditions and a temperature of 30-40°C, pH of 6-8, and air flow rate of 0.5-3 vvm, vi. after completion of bioreactor production, separating the cell-containing biosurfactant liquid from the bioreactor under aseptic conditions and subjecting it to centrifugation, vii. upon collection of cells at the bottom of the centrifuge tubes, separating the resulting cell-free liquid.

8. The method according to claim 7, characterized in that it comprises the process steps of; i. preparing inoculum culture with 2.2% tryptone, 3.2% glucose, 0.6% waste oil, and 1 % yeast extract by volume and sterilizing the prepared culture in autoclave at 121 °C for 20 minutes, ii. adding 1% by volume of Pseudomonas sp. to the inoculum culture left to cool down and preparing it for bioreactor production in a shaking incubator at 35°C for 24 hours at 150 rpm,iii. preparing bioreactor production media containing 2.2% tryptone, 3.2% glucose, 0.6% waste oil, and 1% yeast extract by volume and sterilizing the bioreactor in autoclave at 121 °C for 20 minutes, iv. after the bioreactor has cooled down, connecting the reactor to the system and operating it for 12 hours to stabilize the oxygen, pH, and temperature probes, v. adding inoculum culture developed in a shaking incubator to the bioreactor 12-18 hours later under aseptic conditions and a temperature of 35°C, pH of 7, and air flow rate of 1 vvm, vi. once bioreactor production is complete, separating the cell containing biosurfactant liquid from the bioreactor under aseptic conditions and subjecting it to centrifugation at a temperature of 21 °C at 10.000 g for 15 minutes, vii. upon collection of cells at the bottom of the centrifuge tubes, separating the resulting cell-free liquid.

9. The method according to any one of claims 6-8, characterized in that said Pseudomonas sp. is Pseudomonas aeruginosa.

10. The biosurfactant produced by the method according to any one of claims 6-8.