A bioprocess for simultaneous production of polyhydroxybutyrate and violacein pigment from Himalayan bacterium Iodobacter sp. PCH 194

Abstract

The present disclosure provides a bioprocess for the simultaneous production of polyhydroxybutyrate (PHB) and violacein pigment in a single fermentation using the Himalayan bacterial isolate Iodobacter sp. PCH 194, which has accession number MTCC 25171. PHB has the properties of plasticity, renewable origin, and biodegradability. It can be used for various packaging applications to replace petrochemical-based plastics, thus providing a greener alternative for the environment. Violacein pigment has antioxidant, antitumor, antibacterial, and photoprotective properties and can be used in cosmetic and pharmaceutical applications. The present disclosure provides a method for producing two valuable products, i.e., PHB and violacein pigment, in a single bioprocess, which is therefore economically attractive, time-saving, and commercially viable.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a bioprocess for the simultaneous production of polyhydroxybutyrate (PHB) and violacein pigment in a single fermentation using a novel bacterial isolate Iodobacter sp. PCH 194 (MTCC 25171).The present invention also relates to dual product PHB and violacein pigment produced in a specific medium under a defined set of growth conditions and further to downstream processes developed for the extraction and recovery of PHB and violacein pigment. [Background technology]

[0002] 2. Background of the Invention Most biomolecules and biopolymers are naturally derived to meet some of the needs of humans. PHB is a linear polyester of 3-hydroxybutyrate, which has attracted considerable attention due to its biodegradability and elastomeric properties. Most polymers are derived from petrochemicals. The main concern with the use of petrochemical-based polymers is their non-biodegradable nature, which in addition causes environmental pollution and threats to fossil resources. Approximately 311 million tons / year of plastics are produced worldwide, 90% of which are synthesized from petroleum (Bornscheuer UT. 2016 Feeding on plastic. Science 351: 1154-1155), thereby resulting in millions of tons of CO2 and other harmful emissions. In addition, the non-biodegradable nature of plastics leads to the accumulation of millions of tons on land and in the sea, causing serious threats to food webs and ecosystems. Therefore, there is an urgent need to explore alternative and reliable sources of polymers that can meet the needs of society in an environmentally friendly manner.

[0003] Although many bacteria have been reported to produce PHB and several biotechnological strategies have been used to improve PHB production, the commercial feasibility in terms of economic value still remains a concern. Although many research studies and inventions using low-cost carbon sources to produce PHB and reduce production costs have been found in the past (US Patent No. 7,129,068, US Patent No. 7,666,636), they still do not compete with petrochemical-based plastics (Wang Y et al., 2014 Polyhydroxyalkanoates, challenges and opportunities. Curr. Opin. Biotechnol. 30: 59-65). Moreover, PHB-based bioplastics are relatively cost-intensive to traditional petrochemical-based plastics due to their higher production costs.

[0004] Violacein pigments include the indole-derived, blue-purple-pigmented secondary metabolites violacein and deoxyviolacein, which are produced by several bacteria, such as Chromobacterium and Janthinobacterium. Violacein has various important biological activities, such as antioxidant, antitumor, antibacterial, antiviral, and antiprotozoan activities (Duran N et al., 2016, Advances in Chromobacterium violaceum and properties of violacein- Its main secondary metabolite: A review. Biotechnol. Adv. 34:1030-1045). Violacein and deoxyviolacein are also used in various cosmetics and sunscreens due to their antioxidant, antibacterial, and photoprotective properties.

[0005] Furthermore, in the textile industry, violacein and deoxyviolacein are used as colorants. Violacein and deoxyviolacein are produced by various bacterial strains (Fang M et al. 2015. High crude violacein production from glucose by Escherichia coli engineered with interactive control of tryptophan pathway and violacein biosynthetic pathway. Microb. Cell Fact. 14:8) and there are patents for their production and extraction methods (EP 2545181).

[0006] Therefore, it is undoubtedly clear that although the prior art reports separate processes for the individual production of polyhydroxybutyrate and violacein pigment, none of the prior art provides a commercially viable single bioprocess for the simultaneous production of two valuable products, i.e., polyhydroxybutyrate and violacein pigment.

[0007] Therefore, keeping in view the drawbacks of the prior art reported so far, there is an urgent need to reduce the overall cost of production of polyhydroxybutyrate and violacein pigment by providing an economical and commercially viable single bioprocess for the co-production of both biomolecules, i.e., PHB and violacein pigment, in a single fermentation reaction using glucose as the sole carbon source. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 7,129,068 [Patent Document 2] U.S. Patent No. 7,666,636 [Patent Document 3] European Patent No. 2545181 [Non-patent literature]

[0009] [Non-Patent Document 1] Bornscheuer UT. 2016 Feeding on plastic. Science 351: pp. 1154 - 1155 [Non-Patent Document 2] Wang Y et al., 2014 Polyhydroxyalkanoates, challenges and opportunities. Curr. Opin. Biotechnol. 30: pp. 59 - 65 [Non-Patent Document 3] Duran N et al., 2016, Advances in Chromobacterium violaceum and properties of violacein - Its main secondary metabolite: A review. Biotechnol. Adv. 34: pp. 1030 - 1045 [Non-Patent Document 4] Fang M et al., 2015. High crude violacein production from glucose by Escherichia coli engineered with interactive control of tryptophan pathway and violacein biosynthetic pathway. Microb. Cell Fact. 14: 8 [Non-Patent Document 5] Sathiyanarayanan G et al., 2017. Production and characterization of medium-chain-length polyhydroxyalkanoates copolymer from Arctic psychrotrophic bacterium Pseudomonas sp. PAMC 28620. Int. J. Biol. Macromol. 97: pp. 710 - 720 [Non-Patent Document 6] Kumar V et al., 2018. Bioplastic reservoir of diverse bacterial communities revealed along altitude gradient of Pangi-Chamba trans-Himalayan region. FEMS Microbiol. Lett. 365: fny144 Summary of the Invention [Problem to be solved by the invention]

[0010] Object of the invention Therefore, the main objective of the present invention is to provide a method for the simultaneous production of PHB and violacein pigment in a single fermentation using a novel bacterial strain under a defined set of physiological growth conditions and media, which removes the limitations of the prior art reported so far.

[0011] A second object of the present invention is to provide an isolated and pure bacterial strain of Iodobacter sp. PCH 194 (MTCC 25171).

[0012] The third object of the present invention is to use glucose as the sole carbon source and tryptone as the nitrogen source for the simultaneous production of PHB and violacein pigment. A fourth object of the present invention is to provide a simple method for the separation, extraction and recovery of violacein pigment and PHB from the culture broth. [Means for solving the problem]

[0013] Summary of the Invention In one embodiment of the present disclosure, there is provided a bioprocess for the simultaneous production of polyhydroxybutyrate (PHB) and a blue-purple violacein pigment using an isolated bacterial strain of Iodobacter sp. MTCC 25171, comprising: (i) mixing a loopful of Iodobacter sp. MTCC 25171 in 50 mL of sterile nutrient broth medium; 25171 and incubating at 100-200 rpm for 32-36 hours at a temperature of 20±2°C to obtain a seed culture for inoculating a production medium; (ii) inoculating 3-5% (v / v) of the seed culture obtained in step [i] into a sterile production medium and incubating at 100-200 rpm for 90-100 hours at a temperature of 20±2°C to obtain a biomass; (iii) harvesting the biomass obtained in step [ii] to obtain a pigmented cell pellet by centrifugation or microfiltration; (iv) suspending the pigmented cell pellet obtained in step [iii] in methanol or ethanol in a volume ratio of 1:3 to 1:5 and mixing thoroughly at room temperature for 10-15 minutes, followed by centrifugation to obtain a pigment in the supernatant; and repeating the steps until no purple color remains in the cell pellet, to obtain a colorless cell pellet; (v) harvesting the biomass obtained in step [iv] Concentrating the supernatant using vacuum evaporation to obtain the desired violacein pigment; (vi) suspending the colorless cell pellet obtained in step [iv] in 3-5 volumes of 1:1 trichloromethane and bleach solution followed by overnight incubation under continuous shaking at a temperature ranging from 30-40°C to obtain a mixture; (vii) allowing the mixture obtained in step [vi] to stand for a period of 10-15 minutes, pipetting out the lower organic layer into a test tube and then adding 3-5 volumes of chilled methanol followed by mixing it thoroughly and keeping it at a temperature ranging from -4 to -20°C for a period of 30-60 minutes for precipitation to obtain a reaction mixture; (viii) centrifuging the reaction mixture obtained in step [vii] to collect the pellet as precipitate and remove residual solvent; (ix) drying the pellet obtained in step [viii] and redissolving it in trichloromethane;and (x) repeating steps (vi-viii) and finally collecting polyhydroxybutyrate pellets by evaporating residual solvent.

[0014] In a second aspect of the present disclosure, a bioprocess for the co-production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein, wherein the obtained PHB is useful for the preparation of biodegradable bioplastics, is provided.

[0015] These and other features, aspects, and advantages of the present subject matter will become better understood with reference to the following description and appended claims. This Summary is provided to introduce a selection of concepts in a simplified form. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 depicts a neighbor-joining phylogenetic tree based on 16S rDNA sequences showing the phylogenetic position of Iodobacter species PCH 194 and related bacteria and type strains (also mentioned at the end of the entry). The tree was constructed in MEGA6 with 1000 bootstrappings, the scale bar represents 0.01 substitutions per nucleotide, and the numbers in parentheses are the GenBank accession numbers of the sequences. [Diagram 2] FIG. 13 depicts biomass, glucose utilization, PHB and violacein pigment production profiles of Iodobacter sp. PCH 194 in 1 liter production medium, pH 7.5, at 20° C. and 150 rpm. [Diagram 3] FIG. 13. Simultaneous production of PHB and violacein pigment by Iodobacter sp. PCH 194 in a 7.5 liter operating volume fermentor. [Figure 4]FIG. 13 depicts GC MS analysis of extracted and purified PHB produced by Iodobacter sp. PCH 194: (a) gas chromatogram of methanolysis degraded PHB, (b) mass spectrum of PHB. [Diagram 5] FIG. 1 depicts NMR analysis of PHB produced by Iodobacter sp. PCH 194: (a) proton NMR, (b) 13C NMR. [Figure 6] FIG. 13 depicts violacein dye monitoring using (a) spectrophotometer: visible spectrum of crude violacein in red, green and blue, while the pink line represents the methanol control, (b) UPLC chromatogram of violacein dye at 570 nm, and (c) mass spectrum of peaks: (c1) RT 5.1 consistent with violacein; molecular weight 343.342, MS mass 344.44, and (c2) RT 5.8; molecular weight 327.343, MS mass 328.41 consistent with deoxyviolacein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description of the Invention The invention will now be described in detail with reference to certain preferred and optional embodiments so that its various aspects may be more fully understood and appreciated.

[0018] Definition: For convenience, before further description of the present disclosure, certain terms used in the present specification and examples are detailed herein.These definitions should be read in the light of the remainder of the present disclosure and should be understood as by those skilled in the art.Terms used herein have meanings that are recognized and known to those skilled in the art, however, for convenience and completeness, certain terms and their meanings are provided below.

[0019] The articles "a", "an" and "the" are used to refer to one or to more than one (ie, to at least one) of the grammatical object of the article.

[0020] The terms "comprise" and "comprising" are used in an inclusive, open sense in which additional elements may be included. It is not intended to be interpreted as "consists of only."

[0021] Throughout this specification, unless the context otherwise requires, the word "comprise", as well as variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step, or group of elements or steps, but not the exclusion of any other element or step, or group of elements or steps.

[0022] Ratios, concentrations, amounts, and other numerical data may be expressed in a range format herein. It should be understood that such range formats are used merely for convenience and brevity, and should be interpreted flexibly to include not only the numerical values ​​explicitly recited as range limitations, but also all of the individual numerical values ​​or subranges subsumed within the range as if each numerical value and subrange were explicitly recited. For example, a mixing speed range of 100 rpm to 200 rpm should be interpreted not only to include the explicitly recited limitations of about 100 rpm to about 200 rpm, but also to include subranges such as 120 rpm to 175 rpm, and individual amounts within the stated ranges such as 150 rpm, 186 rpm, and so forth.

[0023] The term "20±2°C" refers to a temperature in the range of 18 to 22°C.

[0024] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure described herein.

[0025] The present disclosure provides a bioprocess for the simultaneous production of two valuable products, namely polyhydroxybutyrate (PHB) and violacein pigment, in a single bioprocess using a novel isolated bacterial strain of Iodobacter sp. PCH 194 [MTCC 25171]. Apart from the scientific and economic benefits, the present disclosure is of high relevance for social and environmental aspects.

[0026] PHB has utility for the production of biodegradable bioplastics. Thus, PHB-based bioplastics can meet the requirements of society in an environmentally friendly manner. In addition, bioplastics made from PHB can significantly contribute to the reduction of pollution caused by plastics in a sustainable and long-term manner. The market for polyhydroxyalkanoates is expected to grow from US$ 73.6 million in 2016 to US$ 93.5 million in 2021, at a CAGR of 4.88%.

[0027] The second product of the biomethod of the present disclosure is violacein pigment, which is a blue-purple colored secondary metabolite containing violacein and deoxyviolacein as major constituents. Violacein has various important biological activities, such as antioxidant, antitumor, antibacterial, and photoprotective properties. Therefore, violacein pigment has applications in the textile, pharmaceutical, and cosmetic industries.

[0028] The present disclosure further provides a pure bacterial strain of Iodobacter sp. PCH 194 [MTCC 25171], wherein colonies of said Iodobacter sp. are purple pigmented, Gram-negative, glucose-fermenting, and grow optimally at a temperature of 20° C.

[0029] In one embodiment of the present disclosure, there is provided a bioprocess for the simultaneous production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using an isolated bacterial strain of Iodobacter sp. MTCC 25171, comprising: i) inoculating a loopful of Iodobacter sp. MTCC 25171 in a sterile nutrient broth medium and incubating at a temperature of 20±2° C. for 32-36 hours at 100-200 rpm to obtain a seed culture for inoculating the production medium; ii) inoculating 3-5% (v / v) of the seed culture obtained in step [i] into a sterile production medium and incubating at a temperature of 20±2° C. for 90-100 hours at 100-200 rpm to obtain biomass; iii) harvesting the biomass obtained in step [ii] to obtain a pigmented cell pellet by centrifugation or microfiltration; iv) suspending the pigmented cell pellet obtained in step [iii] in methanol or ethanol at a volume ratio of 1:3 to 1:5 and mixing thoroughly at room temperature for 10 to 15 minutes, followed by centrifugation to obtain the pigment in the supernatant; and repeating the process until no purple color remains in the cell pellet, to obtain a colorless cell pellet; v) concentrating the supernatant obtained in step [iv] using vacuum evaporation to obtain the desired violacein dye; vi) suspending the colorless cell pellet obtained in step [iv] in 3-5 volumes of 1:1 trichloromethane and bleach solution, followed by overnight incubation at a temperature ranging from 30-40° C. under continuous shaking to obtain a mixture; vii) allowing the mixture obtained in step [vi] to stand for 10-15 minutes, pipetting out the lower organic layer into a test tube, and then adding 3-5 volumes of chilled methanol, followed by thorough mixing and keeping it at a temperature ranging from -4 to -20°C for 30-60 minutes for precipitation to obtain a reaction mixture; viii) centrifuging the reaction mixture obtained in step [vii] to collect the pellet as a precipitate and remove residual solvent; ix) drying the pellets obtained in step [viii] and redissolving them in trichloromethane; and x) Repeating steps (vi-viii) and finally collecting the polyhydroxybutyrate pellets by evaporating the residual solvent. A method is provided, comprising:

[0030] In an embodiment of the present disclosure, a bioprocess for simultaneous production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein, wherein the production medium of the process comprises 4% (w / v) glucose, 0.5% (w / v) tryptone, 0.1% (w / v) NaCl and 25 mM K2HPO4 / KH2PO4, pH 7.5, inoculated with 4% (v / v) seed.

[0031] In one embodiment of the present disclosure, a bioprocess for simultaneous production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein is provided, wherein the duration for the production of PHB and violacein pigment ranges from 36 to 100 hours at a temperature of 20° C. with continuous shaking at 150 rpm. In another embodiment of the present disclosure, the duration for the production process ranges from 36 to 98 hours with continuous shaking at 150 rpm. In yet another embodiment of the present disclosure, the duration for the production process is 96 hours with continuous shaking at 150 rpm.

[0032] In an embodiment of the present disclosure, a bioprocess for simultaneous production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein is provided, wherein the process results in production of pigmented biomass in the range of 7-9 g / L dry cell mass from 1 liter of culture broth after harvesting using centrifugation.

[0033] In one embodiment of the present disclosure, a bioprocess for the simultaneous production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein is provided, which results in the production of pigmented biomass in the range of 13-19 g / L dry cell mass after centrifugation or microfiltration of 7.5 or 10 liter fermenter broth using an Akta Flux microfiltration system (GE Healthcare). The 7.5 culture broth was concentrated using a 0.2 μm hollow fiber cartridge, and the remaining purple pigmented cell slurry was then collected and centrifuged. After centrifugation, both the supernatant and pellet were collected for further extraction of violacein pigment and PHB. The viscous supernatant was treated with 3-5 volumes of methanol or ethanol and stirred using a magnetic stirrer. The mixture was again passed through the Akta flux system or centrifuged to obtain violacein pigment as filtrate or supernatant. In another embodiment of the present disclosure, a bioprocess for simultaneous production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein is provided, in which the cell pellet of the process is treated with methanol, mixed thoroughly at room temperature, centrifuged, and the steps are repeated until the pellet becomes colorless. In yet another embodiment of the present disclosure, the supernatant is simultaneously collected in each step by evaporating in a rotary vacuum evaporator.

[0034] In another embodiment of the present disclosure, a bioprocess for simultaneous production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein is provided, wherein the solvent used for extraction may be selected from the group consisting of methanol, ethanol, or butanol in a ratio ranging from 1:3 to 1:5, and the extraction process is repeated 2-3 times until no additional color remains in the cell pellet. In yet another embodiment of the present disclosure, the crude extract of violacein pigment is concentrated by vacuum evaporator.

[0035] In another embodiment of the present disclosure, a bioprocess for simultaneous production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein is provided, wherein colorless cell pellet is treated with 3-5 volumes of 1:1 trichloromethane and bleach solution at 37° C. overnight under continuous shaking condition.

[0036] In another embodiment of the present disclosure, a bioprocess for simultaneous production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein is provided, wherein the mixture of overnight incubated cell pellet, trichloromethane and bleach solution is allowed to stand for 10-15 minutes, the lower organic layer is pipetted into a test tube or container, chilled methanol in a volume of 1:5-1:10 is added thereto, mixed thoroughly, kept at a temperature ranging from -4°C to -20°C for 30-60 minutes, and centrifuged.

[0037] In another embodiment of the present disclosure, a bioprocess for simultaneous production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein is provided, wherein the pellets are collected, dried, re-dissolved in trichloromethane, the methanol precipitation step is repeated, and finally the pellets of PHB are collected, dried and the residual solvent is evaporated.

[0038] In one embodiment of the present disclosure, a bioprocess for simultaneous production of polyhydroxybutyrate (PHB) and violacein pigment, a blue-purple color, using an isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein is provided, wherein the duration for production of PHB and violacein pigment in a fermenter ranges from 36 to 100 hours while maintaining 20% ​​dissolved oxygen and 150 rpm. In another embodiment of the present disclosure, the duration for production of PHB and violacein pigment in a fermenter is 96 hours. In one embodiment of the present disclosure, a bioprocess for simultaneous production of polyhydroxybutyrate (PHB) and violacein pigment, a blue-purple color, using an isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein is provided, wherein the yield of PHB and violacein pigment obtained from 1 liter of culture broth is within the range of 3 to 5 g / L (>50% of dry cell biomass) and 0.5 to 0.8 g / L, respectively.

[0039] In an embodiment of the present disclosure, a bioprocess for the co-production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein is provided, wherein the yield of PHB and violacein pigment from 7.5 or 10 liters of fermenter broth is in the range of 7-10 g / L (>50% of dry cell biomass) and 0.8-1.5 g / L, respectively.

[0040] In another embodiment of the present disclosure, a bioprocess for simultaneous production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein is provided, wherein PHB and violacein pigment production can be produced by using various concentrations of, but not limited to, 4% (w / v) glucose and 0.5% (w / v) tryptone.

[0041] In yet another embodiment of the present disclosure, PHB and violacein pigment production can be achieved in a variety of organic carbon sources and a variety of inorganic and organic nitrogen sources, including but not limited to glucose and tryptone.

[0042] In another embodiment of the present disclosure, a bioprocess for simultaneous production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein is provided, wherein PHB and violacein pigment production can be achieved in a wider range of pH, temperature, oxygen and rpm conditions, including but not limited to, 20° C. temperature, pH 7.5, 20% dissolved oxygen and 150 rpm.

[0043] In one embodiment of the present disclosure, a bioprocess for simultaneous production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein is provided, wherein the yield of PHB (PHB / carbon source) is in the range of 0.18-0.25 g / g.

[0044] In another embodiment of the present disclosure, the volumetric productivity of PHB is in the range of 0.075 to 0.104 g / L / hr.

[0045] In another embodiment of the present disclosure, the fermentation is carried out at a variable pH range during the fermentation in the context of an initial pH of up to 7.5.

[0046] In another embodiment of the present disclosure, a bioprocess for co-production of polyhydroxybutyrate (PHB) and blue-purple violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein, wherein the obtained PHB has a melting point (Tm) of 178±5°C and a crystallization temperature of 50±5°C is provided.

[0047] In an embodiment of the present disclosure, a bio-method for the simultaneous production of polyhydroxybutyrate (PHB) and blue-violet violacein pigment using an isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein, wherein the resulting PHB is useful for the preparation of biodegradable bioplastics is provided. In an embodiment of the present disclosure, a bio-method for the simultaneous production of polyhydroxybutyrate (PHB) and blue-violet violacein pigment using an isolated bacterial strain of Iodobacter sp. MTCC 25171 disclosed herein, wherein the resulting violacein pigment exhibits antibacterial, anticancer and photoprotective properties is provided. EXAMPLES

[0048] The following examples are given for illustrative purposes and therefore should not be construed as limiting the scope of the present invention.

[0049] Details of the biological resources used in the present invention The bacterial strain Iodobacter sp. PCH 194 was isolated from a muddy sediment sample of a high altitude glacial lake in the Pangi and Chamba regions of the Western Himalayas, Himachal Pradesh, 176316, India. The bacterial isolate PCH194 was identified based on partial 16S rDNA sequencing and phylogenetic analysis with reference strains. The bacterial strain Iodobacter sp. PCH 194 was deposited at MTCC, CSIR-IMTECH, Chandigarh, India under accession number MTCC 25171 on 27 February 2018.

[0050] methodology Isolation and identification of bacterial strains Sediment samples were collected from a high altitude glacial lake in the Pangi-Chamba region of the Western Himalayas and transported to the laboratory at 4°C. Sediment samples were directly plated in duplicate on Antarctic Bacteria Agar (ABM). Plates were incubated at 20°C until colonies appeared and pure bacterial colonies were further streaked on Nutrient Agar (NA). Purple colonies were purified by streaking on NA plates and identified by 16S rDNA sequencing using a DNA ABI 3130XL Genetic Analyzer (Applied Biosystems). Bacterial isolate PCH194 is purple (most intense in the center of the spread colony), Gram-negative, small rod-shaped, cells are straight round-ended rod-shaped, facultative anaerobe, oxidase and catalase positive, glucose fermenting, and can grow from 4 to 28°C with optimal growth at 20°C. Molecular characterization of the partial 16S rDNA sequence showed >99% sequence similarity to Iodobacter fluviatilis ATCC 33051(T), and phylogenetic analysis also showed the closest match to this type strain (Figure 1). Bacterial cultures can be maintained on nutrient agar plates at 4°C with subculturing after 20-30 days, or in glycerol stocks at -80°C with subculturing every 6 months.

[0051] Production of PHB and violacein pigments in 1 liter flasks and fermenters of 7.5 and 10 liter operating volumes The production of PHB and violacein pigments was carried out in flasks at 1 liter working volume using optimized production medium components (4% glucose, 0.5% tryptone, 0.1% NaCl and 25 mM phosphate buffer components), pH 7.5 and production conditions (90-100 hours incubation at 20°C and 150 rpm). For further scale-up, 23.5 liter fermenters with 7.5 and 10 liter working volumes were run using slightly higher medium components (4.25% glucose, 0.55% tryptone, 0.1% NaCl and 25 mM potassium phosphate buffer components in % (w / v)) and the medium pH was set at 7.5. The fermenters were inoculated with 4% (w / v) seed (32-36 h old) and incubated at 20°C for 90-100 h at 150 rpm, with uncontrolled pH and pO2 automatically set at 20%. 1.0 or 2.0 ml of antifoam (Antifoam SE-15, Sigma) was added to the fermenter as needed to limit foam formation in the fermenter.

[0052] Downstream processing of violacein pigment from biomass Culture broth biomass was harvested after 96 h by centrifugation (10,000 g for 10 min at 4–10°C) from 1 liter culture broth and by microfiltration using an Akta Flux system (GE Healthcare, USA) from 7.5 or 10 liter fermenter broth. The harvested pigmented cell pellet was suspended in methanol or ethanol at a ratio of 1:3–1:5, mixed thoroughly at room temperature for a few minutes, and then centrifuged to obtain the pigment in suspension. This process was repeated until no purple color remained in the cell pellet.

[0053] Culture broth from 7.5 or 10 liter fermenters was concentrated using microfiltration in an Akta flux system. The reaction was mixed with 3-5 volumes of methanol and stirred using a magnetic stirrer. This mixture was again passed through the Akta flux system and the violacein pigment was collected as the filtrate. The retentate was again treated with methanol and the process was repeated until the cell pellet was colorless. The extracted violacein pigment solution was concentrated and dried using a rotary vacuum evaporator to obtain the desired bacterial crude violacein pigment.

[0054] Downstream processing of PHB from biomass The remaining colorless cell pellet was suspended in 3-5 volumes of 1:1 trichloromethane and bleach solution and incubated overnight at 37°C under continuous shaking conditions. The mixture was allowed to stand and cool for a few minutes and the lower organic layer was pipetted into separate tubes or reagent bottles. To this, chilled methanol was added (1:5-1:10 by volume), mixed thoroughly, kept at a temperature ranging from -4°C to -20°C for 30-60 minutes and centrifuged. The PHB pellet was dissolved again in trichloromethane and the precipitation process was repeated again. Finally, the PHB pellet was collected, dried to evaporate the residual solvent and quantified.

[0055] Quantification and chemical profiling of polyhydroxyalkanoates and violacein pigments The absorbance of violacein dye was measured at 570 nm and the spectral analysis (300-700 nm) was performed in a multiplate reader (BioTek Instruments, USA). The crude methanol extract was dried in a vacuum rotavapor, weighed and redissolved in HPLC grade methanol for further analysis. A standard curve of violacein dye was prepared by dissolving known amounts in methanol and recording the absorbance at 570 nm. This curve was used for the estimation of violacein dye in further assays. Chemical profiling, quantification and characterization of violacein dye was performed in UPLC-MS.

[0056] Determination of Monomer Composition of Polyhydroxyalkanoates Using GC-MS The composition of PHA monomers was determined by GCMS analysis of the methanolyzed samples on a GCMS-QP2010 (Shimadzu). Digestion and derivatization of purified PHA were performed by using the methods described (Sathiyanarayanan G et al., 2017. Production and characterization of medium-chain-length polyhydroxyalkanoates copolymer from Arctic psychrotrophic bacterium Pseudomonas sp. PAMC 28620. Int. J. Biol. Macromol. 97: 710-720; Kumar V et al., 2018. Bioplastic reservoir of diverse bacterial communities revealed along altitude gradient of Pangi-Chamba trans-Himalayan region. FEMS Microbiol. Lett. 365: fny144). PHA monomers were identified by searching the NIST / EPA / NIH library and comparing the corresponding mass ions of each peak.

[0057] Characterization NMR analysis For NMR analysis, the purified PHA was 1 H) and carbon ( 13 C) For determination of chemical structure by using NMR, the compounds were dissolved in deuterated chloroform (CDCl3). NMR spectra were recorded using a Bruker Avance III 600 (Bruker Co., Fallanden, Switzerland). 1 H and 13 C spectra were obtained at room temperature at 500 and 150 MHz, respectively. Chemical shifts such as resonance signals (δ) were given in ppm relative to prominent signals of CDCl3 as internal standard (1 H NMR: 7.26 ppm; 13 C NMR: 77.42).

[0058] Preparation of PHB bioplastic film and analysis of its physical properties Films of PHB were prepared by dissolving purified PHB in trichloromethane and evaporating the solvent. Thermal properties such as melting point (Tm) and crystallization temperature (Tc) were calculated using outsourced facilities provided by Nation Physical Laboratory, New Delhi.

[0059] Isolation, identification and phylogeny of PCH194 The bacterium is purple, gram-negative, facultative anaerobe, sugar-fermenting and can grow between 4 and 30°C, with optimum growth at 20°C.

[0060] Bacterial cultures can be maintained on nutrient agar plates at 4° C. with subculturing every 15-20 days, or in glycerol stocks at −80° C. with subculturing every 6 months.

[0061] Molecular characterization of the partial 16S rDNA sequence showed >99% sequence similarity to Iodobacter fluviatilis ATCC 33051(T), and phylogenetic analysis also showed the closest match to this type strain (Figure 1). Iodobacter species PCH194 is a psychrophilic bacterium and showed the potential for PHB and violacein pigment production.

[0062] Growth Profile Iodobacter sp. PCH194 showed good biomass production at 20° C. This bacterium has the ability to synthesize PHB and violacein pigments.

[0063] The effect of various carbon sources on growth and PHB production revealed that glucose was the preferred energy source, resulting in the most biomass and PHB accumulation (>50%). Fructose and maltose also performed well for growth and PHB production, but were relatively underutilized compared to glucose. Glycerol, lactose, sucrose, trisodium citrate, and potassium sodium tartrate were not utilized efficiently by the bacteria, resulting in lower biomass and PHB production.

[0064] Among various nitrogen sources, tryptone was found to be a very good nitrogen source for the synthesis of both PHB and violacein pigment. Other nitrogen sources such as peptone, yeast extract, etc. may also be used, but may result in lower PHB yields. Among inorganic nitrogen sources, NH4Cl and NH4SO4 showed good biomass and relatively low production of both PHB and violacein pigment (statement not clear).

[0065] Typical growth curves and fermentation profiles shown in Figure 2 revealed that the production of PHB and purple pigment started after 24 h and steadily increased with cell mass, reaching the highest production in the stationary phase.

[0066] Production and extraction of PHB and violacein pigments under 1-liter flask conditions The production of PHB and violacein was carried out at 1 liter scale in shake flasks, resulting in the production of 7-9 g / L of dry cell biomass in 90-100 hours of incubation, with the cell density remaining nearly constant up to 144 hours. Cells harvested after 96 hours of incubation were used for the extraction of PHB and violacein pigments discussed. The yields of PHB and crude violacein pigment from 1 liter culture of the method after 96 hours are in the range of 3-5 g / L (>50% of dry cell biomass) and 0.5-0.8 g / L, respectively (Figure 2).

[0067] Production and extraction of PHB and violacein pigments in 7.5 or 10 liter fermenters In fermenters with an operating volume of 7.5 or 10 liters using a constant DO (20%), the OD of the cells was 600 reached ≥15 in 90-100 h of fermentation, and the dry cell mass was 16-19 g / L. After the discussed biomass harvesting and extraction methods, the PHB and violacein pigment yields of the method were found to be in the range of 7-10 g / L (>50% of dry cell biomass) and 0.8-1.5 g / L, respectively (Figure 3). The presence of O2 is necessary for the production of violacein, however, the bacteria can produce PHB in facultative anaerobic conditions. Therefore, pO2 levels were maintained at 20% for the simultaneous production of PHB and violacein pigment using Iodobacter sp. PCH 194 bacteria in the method.

[0068] Analysis of PHB, PHB-based bioplastic films, and violacein pigment The extracted PHB was analyzed by GC-MS, which revealed 3-hydroxybutyrate as the monomer (Figure 4), and NMR (C and H) confirmed that the polymer was PHB (Figure 5). Furthermore, thermal property analysis showed a melting point (Tm) of 178 ± 5 °C and a crystallization temperature (Tc) of 50 ± 5 °C (Table 1). These properties are comparable to PHB produced from other bacteria and reported previously.

[0069] Methanol extracted crude violacein pigment analyzed using UPLC-MS showed a purity of >70% of the sample and the presence of >40% violacein and deoxyviolacein in the total mixture with mass fragmentation patterns consistent with molecular weights of 343.342 g / mol for violacein and 327.343 g / mol for deoxyviolacein, respectively (Figure 6).

[0070] [Table 1]

[0071] Example 1 Production of PHB in a fermenter with an operating volume of 7.5 liters without O2 control A loopful of Iodobacter sp. MTCC 25171 was inoculated in 50 mL of sterile nutrient broth medium and incubated at 100-200 rpm for 32-36 h at a temperature of 20 ± 2 °C to obtain the seed culture for inoculating the production medium. 4% (v / v) seed (32 h old) was inoculated into 7.5 liters of sterile production medium containing 4% (w / v) glucose, 0.5% (w / v) tryptone, 0.1% (w / v) NaCl and 25 mM K2HPO4 / KH2PO4 at pH 7.5. Fermentation was carried out at 20 °C for 96 h at 100 rpm without oxygen control. Biomass was harvested after 96 h to obtain cell pellets by centrifugation. The cell pellet was suspended in 3 volumes of 1:1 trichloromethane and bleach solution and incubated overnight at 37 °C under continuous shaking conditions. The mixture was kept for a few minutes, the lower organic layer was pipetted into a tube, and another 3-5 volumes of chilled methanol were added thereto, mixed thoroughly, and kept at -20°C for a few minutes for precipitation. The reaction mixture was centrifuged to collect the precipitate, which was dissolved in a minimum amount of trichloromethane and precipitated with 3 volumes of methanol used. The reaction mixture was then centrifuged to collect the precipitated PHB, and residual solvent was removed by drying. The dried PHB was weighed and analyzed. The method yielded a biomass of 9 g / L dry cell mass and PHB of 5 g / L.

[0072] Example 2 Production of PHB and violacein pigments in a fermenter with 7.5 litre operating volume using 20% ​​dissolved O2 4% (v / v) seed (36 h old) obtained by the method described in Example 1 was inoculated into 7.5 liters of sterile production medium containing 4.25% (w / v) glucose, 0.55% (w / v) tryptone, 0.1% (w / v) NaCl and 25 mM K2HPO4 / KH2PO4 at pH 7.5. Fermentation was carried out at 20°C for 96 h at 150 rpm with automatic control of pO2 of 20%. Biomass was harvested after 96 h to obtain cell pellets by centrifugation. The method resulted in the production of a pigmented biomass (approximately 19 g / L dry cell mass) after harvest. Harvesting was carried out using microfiltration of the broth using an Akta Flux system (GE Healthcare) with a 0.2 μm hollow fiber cartridge. The purple pigmented cell slurry was collected and centrifuged. After centrifugation, both the supernatant and pellet were collected for further extraction of violacein dye and polyhydroxybutyrate. The supernatant was very viscous and therefore was treated with 3 volumes of methanol and stirred using a magnetic stirrer. The mixture was centrifuged again and the violacein dye was collected as the supernatant. The cell pellet was treated with 5 volumes of methanol, mixed thoroughly at room temperature, incubated for 10 minutes, followed by centrifugation. This process was repeated twice and the pellet became colorless. The collected violacein dye solution was concentrated in a rotary vacuum evaporator. The remaining cell pellet was mixed with 5 volumes of 1:1 trichloromethane and bleach solution and incubated at 37°C overnight under continuous shaking conditions. The mixture was kept for a few minutes and the lower organic layer was pipetted. 5 volumes of chilled methanol was added to the pipetted organic solution, mixed thoroughly and kept at -20°C for a few minutes for precipitation. The reaction mixture was centrifuged, the precipitate collected, washed with two volumes of methanol, and residual solvent was removed by drying. The dried PHB was weighed and analyzed. The method yielded approximately 19 g / L dry cell mass biomass, 10 g / L PHB, and 1.5-1.8 g / L violacein pigment.

[0073] Example 3 Production of PHB and violacein pigments in a fermenter with 10 litre operating volume using 20% ​​dissolved O2 4% (v / v) seed (36 h old) obtained by the method described in Example 1 was inoculated into 10 liters of sterile production medium containing 4.0% (w / v) glucose, 0.5% (w / v) tryptone, 0.1% (w / v) NaCl and 25 mM K2HPO4 / KH2PO4 at pH 7.5. Fermentation was carried out at 20°C for 96 h at 150 rpm with automatic control of pO2 of 20%. Biomass was harvested after 96 h to obtain cell pellets by centrifugation. The method resulted in the production of colored biomass after harvest. Harvesting was carried out using microfiltration of the broth using an Akta Flux system (GE Healthcare) with 0.2 μm hollow fiber cartridges. The purple pigmented cell pellet was collected, treated with 5 volumes of methanol and stirred using a magnetic stirrer. The mixture was again passed through an Akta Flux microfiltration system. The purple filtrate was collected and concentrated in a rotary vacuum evaporator. The remaining colorless cell pellet was mixed with 3 volumes of 1:1 trichloromethane and bleach solution and incubated at 37°C overnight under continuous shaking conditions. The mixture was kept for a few minutes and the lower organic layer was pipetted. 5 volumes of chilled methanol was added to the pipetted organic solution, mixed thoroughly and kept at -20°C for a few minutes for precipitation. The reaction mixture was centrifuged to collect the precipitate, washed with 2 volumes of methanol and residual solvent was removed by drying. The dried PHB was weighed and analyzed. The method yields a biomass of 13 g / L dry cell mass, PHB of 7 g / L, and violacein pigment of 0.8-1.2 g / L.

[0074] Advantages of the invention The process developed produces two valuable products, namely polyhydroxybutyrate and violacein pigment, in a single fermentation followed by downstream processing to obtain the individual products.

[0075] Higher production of both products, i.e., PHB and violacein pigment, in a single process.

[0076] A simple method for the separation, extraction, and recovery of violacein pigment and PHB from culture broth.

[0077] The process is carried out without pH control.

[0078] The production medium is very simple.

[0079] The process produces 7-10 g / L PHB (>50% of dry cell biomass) and 0.8-1.5 g / L violacein pigment from fermentors with an operating volume of 7.5 or 10 liters.

[0080] The bioprocess is economically attractive because the process produces two products in a single fermentation.

[0081] Violacein obtained from the method of the present disclosure has been established for its known important biological activities, such as antioxidant, antitumor, antibacterial, antiviral and antiprotozoan. PHB-based biopolymers obtained from the method of the present disclosure are useful for synthesizing biodegradable bioplastics.

[0082] The products of the present disclosure find enormous application in the chemical, cosmetic, biotechnology, bioplastics, and biomaterials sectors.

Claims

1. A bioprocess for the simultaneous production of polyhydroxybutyrate (PHB) and violet-blue violacein pigment using an isolated bacterial strain of Iodobacter sp. MTCC 25171, comprising: i) inoculating a loopful of Iodobacter sp. MTCC 25171 in 50 mL of sterile nutrient broth medium and incubating at a temperature of 20±2° C. for 32-36 hours at 100-200 rpm to obtain a seed culture for inoculating the production medium; ii) inoculating 3-5% (v / v) of the seed culture obtained in step [i] into a sterile production medium and incubating at a temperature of 20±2° C. for 36-100 hours at 100-200 rpm to obtain biomass; iii) harvesting the biomass obtained in step [ii] to obtain a pigmented cell pellet by centrifugation or microfiltration; iv) suspending the pigmented cell pellet obtained in step [iii] in methanol or ethanol at a volume ratio of 1:3 to 1:5 and mixing at room temperature for 10 to 15 minutes, followed by centrifugation to obtain the pigment in the supernatant; and repeating the process until no purple color remains in the cell pellet, to obtain a colorless cell pellet; v) concentrating the supernatant obtained in step [iv] using vacuum evaporation to obtain the desired violacein dye; vi) suspending the colorless cell pellet obtained in step [iv] in 3-5 volumes of 1:1 trichloromethane and bleach solution, followed by overnight incubation at a temperature ranging from 30-40° C. under continuous shaking to obtain a mixture; vii) allowing the mixture obtained in step [vi] to stand for 10-15 minutes, pipetting out the lower organic layer into a test tube, and then adding 3-5 volumes of chilled methanol, followed by mixing it and keeping it at a temperature ranging from -4 to -20°C for 30-60 minutes for precipitation to obtain a reaction mixture; viii) centrifuging the reaction mixture obtained in step [vii] to collect the pellet as a precipitate and remove residual solvent; ix) drying the pellets obtained in step [viii] and redissolving them in trichloromethane; and x) Repeating steps (vi-viii) and finally collecting the polyhydroxybutyrate pellets by evaporating the residual solvent. A method comprising:

2. The method described in claim 1, wherein the incubation in steps [i] and / or [ii] is performed at 20°C and 150 rpm.

3. The method described in claim 1, wherein the production medium in steps [i] and / or [ii] maintains a dissolved oxygen concentration of 20%.

4. The method according to claim 1, wherein the yields of PHB and violacein pigments are within the ranges of 3 to 10 g / L and 0.5 to 1.5 g / L, respectively.

Images