PUTIDA MAO12

FIELD OF THE INVENTION

The present invention relates to the discovery of a novel strain of Pseudomonas putida that is capable of producing high levels of poly B-hydroxybutrate (PHB) on inexpensive carbon sources. PHB is becoming increasingly important as a non-toxic, environmentally- friendly alternative to petroleum-based plastics.

BACKGROUND OF THE INVENTION

Poly-beta-hydroxybutyrate (PHB) was first discovered in the 1920s, but the work was largely unnoticed. Lemoigne, M., "Produits de Dehydration et de Polymerisation de L'acide β-Oxobutyrique," Bull. Soc. Chim. Biol., Vol. 8 pp. 770-82 (1926). It is a biocompatible thermoplastic material of the polyester class known as polyhydroxyalkanoates (PHAs). PHBs are produced intracellularly in certain bacterial genera such as Azotobacter and Bacillus. The most active genera in PHB production are Azotobacter, Ralstonia, Bacillus, Pseudomonas and Alicaligens eutrophs See, Singh and Parmar, "Isolation and Characterization of Two Novel Polyhydroxybutrate (PHB)-Producing Bacteria," African Journal of Biotechnology, Vol. 10, Issue 24, pp. 4907-4919 (2011). The PHB accumulates in inclusion bodies in the cytoplasm and are visible as bright granules under light microscopy.

PHB has unique properties such as UV resistance, resistance to hydrolytic degradation, insolubilty in water, oxygen permeability, solubility in chloroform, and poor resistance to acids, bases, and chlorinated organic materials. PHB is generally considered to be a non-toxic biodegradable polymer that may in be used in some instances as a substitute for plastic. PHBs have numerous applications in medicine (e.g., dissolvable sutures) and agriculture. PHBs have been used to make a wide variety of products such as disposable containers and bags as well as packing materials.

Because petroleum- based plastics do not decompose they have become a tremendous waste management problem. Plastics now pollute landfills and waterways, including the oceans, thus endangering both wildlife and humans. Approximately 300 million tons of plastic are produced each year and only about 10% is recycled. Worldwatch Institute, April 28, 2017. Thus, developing inexpensive biodegradable substitutes is important both for living "green" in today's world and reducing oil consumption.

The microorganism of the present invention is capable of producing PHB even when grown on low cost renewable carbon sources. This offers a tremendous cost advantage over industrial production of both PHB and plastic. The PHB produced by the invention is nontoxic, biocompatible and can be degraded by microbes.

The present invention aims to optimize the effect of various environmental conditions on PHB production by the newly-discovered isolate, Pseudomonas putida strain MA012.

SUMMARY OF THE INVENTION

The present invention provides a biologically pure strain of the genus Pseudomonas that is capable of producing high levels of PHB. The bacteria is referred to as Pseudomonas putida strain MAO 12. It was isolated from waste water collected in Saudi Arabia. The strain MAO 12 exhibits sustained growth and PHB production on a variety of inexpensive carbon sources such as corn steep, molasses, chitin, whey and starch, in the presence of 10% glucose.

Plastics have become a grave environmental threat, filling our landfills and waterways and endangering wildlife. The methods of the present invention help to solve this problem by providing an inexpensive means of producing high levels of a biodegradable, biocompatible plastic, PHB. The present methods relate to the growing of a new microbial strain, MAO 12, on renewable carbon sources.

MAO 12 has been deposited at the Culture Collection of Switzerland AG (CCOS) in accordance with the Budapest Treaty. Pseudomonas putida strain MA012 CCOS 1151.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates the growth and production of PHB at different incubation temperatures by MA012. [♦] shows cell dry weight in g/1. [■] depicts %PHB.

FIG.2 demonstrates the effect of different pH values on growth and PHB

production of MA012. [♦] represents cell dry weight in g/1. [■] depicts %PHB.

FIG.3 demonstrates MAO 12 growth and production at different incubation times. [♦] represents cell dry weight in g/1. [■] depicts %PHB.

FIG.4 demonstrates MA012 growth and production of PHB using different carbon sources. [♦] represents cell dry weight in g/1. [■] depicts %PHB.

FIG.5 demonstrates MA012 growth and production of PHB using various waste products as the carbon source. [♦] represents cell dry weight in g/1. [■] depicts %PHB.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

EXAMPLE 1: Bacterial Isolation and Screening for PHB

Samples were collected from soil and waste water at a date palm farm and a waste water treatment station, respectively. Approximately, 200 g samples of soil were collected in sterile plastic bags and 200 ml samples of the waste water were collected in sterile glasses. The soil samples were spread on sterile filter paper and air dried. The dried samples were ground, sieved and rehydrated with water at varying concentrations.

Approximately 0.5 ml of the waste water and reconstituted soil samples were spread on the nutrient agar plates containing g/1: peptone 2 g, yeast extract 2 g, NaCl 1 g, Agar 20 g and 1 % glucose and incubated at 37° C for 24 hours.

The nutrient agar plates were screened using Sudan Black. Panigrahi and Badveli, "Screening, Isolation and Quantification of PHB-Producing Soil Bacteria," International J. Engineering Science, Vol. 2, Issue 9, pp. 1-6 (2013). The growth was covered with 0.02% Sudan Black Dye in ethanol for 30 min then washed with 96% ethanol to remove excess stain.

Out of 20 isolates, six (30%) showed the high PHB production as determined by the very dark black color of the bacterial colonies after washing. Eight (40%) showed moderate PHB production as evidenced by the pale black color of the colonies and six (30%) showed no production as visualized by a gray color.

Six isolates with the highest level of PHB production were selected and grown in nutrient broth containing 10% glucose at 30° C, shaking at 120 rpm, for 2 days. Cell growth and PHB production are shown in Table 1. The isolate MAO 12 had the highest level of PHB production and was selected for further study. The isolate was a Gram negative bacillus obtained from waste water. PHB production for MAO 12 was 0.0S g/1 and the accumulation percentage was 37% of the cell dry weight.

Table 1. Growth and PHB detection and production on either Nutrient agar or in Nutrient Broth containing 10% glucose.

The MAO 12 bacillus was further characterized and found to have no capsule or spore, was positive in the catalase test and negative for ¾S and indole production. Urea, gelatin and starch hydrolysis were also negative. Fermentation of sugars to acids (Methyl Red Test) was negative.

Table 2. Morphological and physiological characters of the selected bacterial isolate MA012

Based on the morphological and physiological characteristics, the isolate was identified as Pseudomonas putida strain MAO 12.

EXAMPLE 2: Quantification of PHB production

Sudan Black positive isolates were grown in 250 ml Erlenmeyer flasks containing 50 ml of nutrient broth with 10% glucose. Each flask was inoculated with 2 ml of medium containing 4x10 ⁶ CFU/ml. The cell cultures were grown at 37° C for 2 hr. then centrifuged at 10,000 rpm for 10 min. The pellets were washed with acetone and ethanol (1:1 V/V), re- suspended into 4% of sodium hypochlorite solution, incubated for 30 min at room temperature and centrifuged. The pellets were washed with equal volume of acetone and ethanol and dissolved in hot chloroform. Following centrifugation at 10,000 rpm for 10 min., 10 ml of concentrated sulphuric acid was added and the presence of crotonic acid was measured at 235 nm using a spectrophotometer. Bonartseva and Myshkina,"The Biodegradation of Poly-B-Hydroxybutyrate (PHB) by a Model Soil Community" The Effect of Cultivation Conditions on the Degradation Rate and the Physicochemical Characteristics of PHB," Microbiology, Vol. 71, Issue 2, pp.258-263 (1985), Aly et al., "Production of Biodegradable Plastic by Filamentous Bacteria Isolated from Saudi Arabia," J. of Food, Agriculture Environment, Vol. 9, Issue 1, pp. 751-756 (2011). Sulfuric acid was used as the control.

In this study, the PHB content differed from one bacteria to another. MAO 12 exhibited the maximum percentage of PHB at 37.0% of dry cell weight. Figure 4. This level of PHB production falls within the low range reported for the genus Azotobacter which is said to be from 35 to 50% per cell dry weight. See, Stockdale, H. et al.,

"Occurrence of Poly-B-Hydroxybutyrate in the Azotobacteriaceae," J. BacterioL, Vol. 95, Issue 5, pp. 1798-1803 (1968). However it is significantly greater than PHB productivity found in Rhizobium spp. 3173 (1.38%). Mercan, N., "Production of Poly-B- Hydroxybutyrate (PHB) by Some Rhizobium Bacteria," Turk. J. Biol., Vol. 26, pp.215-19 (2002).

EXAMPLE 3: Factors affecting PHB production:

The conditions for optimal PHB production by strain MAO 12 was determined by varying the growth factors. The effect of incubation temperature (25, 30, 35, 37, 40 and 45° C), pH (5.0, 6.0, 7.0, 7.5, 8.0 and 8.5) and incubation time (1, 2, 3 and 4 days) on PHB production was determined by growing the isolates in 500 ml conical flasks containing 100 ml nutrient broth with 10% glucose at 120 rpm. Growth (dry weight, g/1) and % of PHB were measured.

Maximum PHB production was achieved by growing strain MA012 in nutrient broth containing 10% glucose at 35° C and pH 7.5 See Figures 1 and 2. Production peaked on day 2 of growth while shaking at 120 rpm. See Figure 3.

Similarly, the effect of different carbon sources at a 10% concentration (glucose, sucrose, maltose, fructose, and cellulose) on PHB production was determined. Here, the bacterial cultures were grown in 250 ml flasks containing 50 ml nutrient broth with different carbon sources. Flasks were inoculated with 2 ml of culture (4x10 ⁶

microorganisms) and incubated at 35° C for 2 days while shaking at 120 rpm. PHB was quantified spectrophotometrically. Law and Slepecky, "Assay of Poly B-Hydroxybutyric Acid Production," J. BacterioL, Vol. 82, pp. 33-36 (1961).

Maximum PHB production in MAO 12 was obtained when grown in nutrient broth containing 10% glucose. Levels of PHB production decreased when grown in fructose, followed by sucrose, maltose, cellulose and finally starch. See Figure 4.

The effect of whey, chitin, molasses, corn steep and starch at a concentration of 10 g /l, was also determined. Cultures were grown in minimal media (Himedia) containing 10% glucose, disodium phosphate (7.9 g/1), potassium phosphate (3.0 g/1), sodium chloride (0.5 g/1) and ammonium chloride (1.0 g/1). atpH 6.8±0.2.

At the end of the growth period, maximum PHB production for MAO 12 was obtained by growing in corn steep, followed by molasses, chitin, whey and starch for 3 days. See Figure 5. Thus, MAO 12 can produce PHB in large scale even when grown on inexpensive, renewable waste products.