CLIAMS:We Claim:
1. Novel non-clostridia bacterial strain of Providencia species having Accession No. MCC0049 that can grow under both aerobic and anaerobic conditions.

2. The novel strain as claimed in claim 1 for producing biofuels.

3. The novel strain as claimed in claims 1-2 for producing biofuels under both aerobic and anaerobic conditions.

4. A process of producing biofuel, said process comprising the steps of:
(a) growing the strain of Providencia species MCC0049 in a culture medium under aerobic or anaerobic conditions at a temperature of 30-35°C at a pH of 5-8 for about 12-24 hours; and
(b) obtaining biofuel.

5. The process as claimed in claim 4, wherein for the aerobic and anaerobic conditions culture medium is selected from T6 medium, LB medium, TSB Medium (TSB) or Reinforced Clostridial Medium.

6. The process as claimed in claims 4-5, wherein the biofuel obtained is butanol, ethanol and acetone or mixture thereof.

7. The process as claimed in claims 4-6, wherein the biofuel obtained is in the range of 2-4 g/L.

8. The process as claimed in claims 4-7, wherein the biofuel is butanol having a concentration range of 2-4 g/L.

9. The process as claimed in claim 4, wherein the step (a) the pH is 7.

10. The process as claimed in claim 4, wherein the step (a) the temperature is 32°C.

11. The novel strain Providencia species MCC0049 for production of butanol biofuel.

12. A method acclimatization of novel strain of Providencia species strain MCC0049, said method comprising the steps of:
(a) isolating the Providencia species strain MCC0049 from the soil samples;
(b) growing the Providencia species strain MCC0049 in aerobic or anaerobic culture medium at the temperature of 25-37°C at a pH of 5-8; and
(c) obtaining acclimatized pure colonies of Providencia species MCC0049.

13. The method as claimed in claim 12, wherein the aerobic and anaerobic culture medium is selected from LB medium, TSB medium, Thioglycolate medium or reinforced clostridial medium.
14. The method as claimed in claim 12, wherein the acclimatized Providencia species strain MCC0049 is useful for producing biofuels.

15. The method as claimed in claim 12, wherein the acclimatized Providencia species strain MCC0049 is useful for producing butanol.
,TagSPECI:FIELD OF THE INVENTION
The present invention relates to a novel non-clostridial bacterial strain for production of biofuels. The present invention also relates to the process for production of biofuels using novel non-clostridial bacterial strain.

BACKGROUND
Increasing cost of fuel, environmental concerns over pollution and awareness of global warming has drawn attention towards the production of biofuels from lignocellulosic biomass. The biological process for biofuel (bioethanol) production from sugar or starch based feedstock via fermentations has been already commercialized. Renewable substrates such as lignocellulosic biomass are now being considered to be of great potential due to their relative abundance, for use as substrates for biofuel production. Similarly, the production of biobutanol as a biofuel has gained considerable attention due to its higher energy density (29MJ/L), less hygroscopic and less corrosive nature when compared to ethanol.

Renewable 1-butanol is produced from the fermentation of carbohydrates in a process often referred to as the ABE fermentation, after its major chemical products: acetone, butanol and ethanol. The ABE fermentation is a proven industrial process that uses solventogenic clostridia to convert sugars or starches into solvents. The fermentation occurs in two stages; the first is a growth stage in which acetic and butyric acids are produced and the second stage is characterized by acid re-assimilation into ABE solvents. During this stage, growth slows, the cells accumulate granulose and form endospores.

Conventionally butanol was produced by bacterial strain Clostridium acetobutylicum in 3:6:1 ratio (Acetone: Butanol: Ethanol). Clostridium acetobutylicum is an obligate anaerobe and is tolerant to 1.5-1.8% butanol. Because of the above mentioned two properties of Clostridium it is industrially difficult to maintain the strain and it is not feasible to produce high amounts of butanol.

Further, synthetic biology has recently been used to introduce biosynthetic capacity for butanol into non-natural hosts. The choice between using or engineering natural function versus importing biosynthetic function has been a difficult one. Commonly used host strains include Escherichia coli and Saccharomyces cerevisiae that are relatively easy to manipulate genetically but do not tolerate more than 2% 1-butanol. In addition, these strains do not display broad substrate ranges and cannot compete with natural or engineered clostridia for the production of 1-butanol from a broad range substrates including pentose sugars and sugars derived from cellulosic feedstocks.

Thus to overcome the limitation of existing technologies, there is a requirement for development of microbial strains other than Clostridia that can produce biofuel (butanol) under aerobic condition and have high tolerance for it.

SUMMARY OF THE INVENTION
Accordingly, the main embodiment of the present invention provides for a novel non-clostridia bacterial strain of Providencia species having Accession No. MCC0049 that can grow under both aerobic and anaerobic conditions.
Another embodiment of the present invention provides for a novel strain as herein described for producing biofuels.
Another embodiment of the present invention relates to a novel strain as herein described for producing biofuels under both aerobic and anaerobic conditions.
Yet another embodiment of the present invention provides for a process of producing biofuel, said process comprising the steps of:
(a) growing the strain of Providencia species MCC0049 in a culture medium under aerobic or anaerobic conditions at a temperature of 30-35°C at a pH of 5-8 for about 12-24 hours; and
(b) obtaining biofuel.
Another embodiment of the present invention provides for a novel strain Providencia species MCC0049 for production of butanol biofuel.
Another embodiment of the present invention provides for a method acclimatization of novel strain of Providencia species strain MCC0049, said method comprising the steps of:
(a) isolating the Providencia species strain MCC0049 from the soil samples;
(b) growing the Providencia species strain MCC0049 in an aerobic or anaerobic culture medium at the temperature of 25-37°C at a pH range of 5-8; and
(c) obtaining acclimatized pure colonies of Providencia species MCC0049.
Another embodiment of the present invention provides for a method of acclimatization as herein described, wherein the aerobic and anaerobic culture medium is selected from LB medium, TSB medium, Thioglycolate medium or reinforced clostridial medium and also T6 medium.
Another embodiment of the present invention provides for a method of acclimatization Providencia species strain MCC0049, wherein the acclimatized Providencia species strain MCC0049 is useful for producing biofuels.
Another embodiment of the present invention provides for a method of acclimatization of Providencia species strain MCC0049, wherein the acclimatized Providencia species strain MCC0049 is useful for producing butanol.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1a: Chromatogram of Providencia K5WT in LB medium with 1% glucose and 1% urea under aerobic conditions
Figure 1b: Standard butanol chromatogram to compare the about chromatogram
Figure 1c: Chromatogram of Providencia K5WT in LB medium with 1% glucose and 1% urea under aerobic conditions.
Figure 1d: Butanol production by Providencia strain K5 with respect to time (in hrs) under anaerobic and aerobic conditions.
Figure 2a: Chromatogram of butanol production by Providencia rettgeri MCC0049.
Figure 2b: Chromatogram of standard butanol to compare butanol production of Providencia rettgeri MCC0049 strain with.
Figure 3a: Chromatogram of butanol production by Clostridium acetobutylicum.
Figure 3b: Chromatogram of standard butanol to compare with butanol produced by Clostridium acetobutylicum.
Figures 4 (a, b and c): Chromatograms depicting ethanol and acetone productions.
Figure 5: Butanol tolerance curve in various concentrations of n-butanol (w/v).
Figure 6 (a, b): Xylose and glycerol utilizations curves of Providencia rettgeri without and with 1% urea.
Figure 7 (a-e): Standard curves of various solvents.
Figure 8: Field emission scanning electron microscopic pictures of Providencia rettgeri.

DETAILED DESCRIPTION
While the invention is susceptible to various modifications and/or alternative processes and/or compositions, specific embodiment thereof has been shown by way of example in the drawings, graphs and tables and will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular processes and/or compositions disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the spirit and the scope of the invention as defined by the appended claims. Before the present methods and the products are described, it is to be understood that this invention is not limited to particular method, product and experimental conditions described; as such methods and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting.
The graphs, tables, figures and protocols have been represented where appropriate by conventional representations in the drawings, showing only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.
The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.
The terms “comprises”, “comprising”, or any other variations thereof, are intended to cover a non-exclusive inclusion, such that one or more processes or composition/s or systems or methods proceeded by “comprises... a” does not, without more constraints, preclude the existence of other processes, sub-processes, composition, sub-compositions, minor or major compositions or other elements or other structures or additional processes or compositions or additional elements or additional features or additional characteristics or additional attributes.

The terms, “alone or in combination” or any other variations thereof, are intended to described and/or cover a non-exclusive inclusion, wherein the molecules or the oligonucleotides exist individually or together with any one or all of the other oligonucleotides.
Definitions:
In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:
It must be noted that, as used in the specification/description and the appended claims and examples, the singular forms “a”, “an” and “the” may include plural referents unless the context clearly dictates otherwise.

Ranges may be expressed herein as from “about” one particular value, and/or “to about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about”, it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

As used herein, the term, “Biomass”, in the context of the present invention means a renewable energy resource derived from the carbonaceous waste of particularly by-products from the timber industry, agricultural crops, raw material from the forest, major parts of household waste and wood. Particularly it is the plants or plant-based materials which is not used for food or feed, and are specifically includes lignocellulosic biomass such as lignocellulosic hydrolysate, etc.

As used herein, the terms, “Non-Clostridial bacterial strain or Non-Clostridial bacteria or bacterial belonging to Non-Clostridial bacterial forms”, in the context of the present invention means refers to bacterial strain belonging to genus Providencia, particularly Providencia rettgeri. The terms “Non-Clostridial bacterial strain or Non-Clostridial bacteria or bacteria belong to Non-Clostridial bacterial forms”, as used in the context of the present invention have been used interchangeably and are meant to have the same definition, meaning and functions.

As used herein, the term, “Biofuel”, in the context of the present invention means is a fuel that is produced through contemporary biological processes, such as agriculture and anaerobic digestion. In context of the present invention the biofuels are produced by the anaerobic breakdown of Biomass by Providencia rettgeri. Thus in context of the present invention biofuels means Biobutanol (n-butanol, isobutanol, 2-butanol and other isoforms of butanol, ethanol and higher forms of alcohols such as but not limited to 3-methyl, 1-butanol, propanol, hexanol, etc.).

As used herein, the term, “Synthetic Media”, in the context of the present invention means artificial laboratory prepared media or commercially available artificial laboratory media, which contains defined nutrient constituents that can be used of reconstituted in a precise reproducible manner.

As used herein, the term, “Natural Media”, in the context of the present invention means nutrient constituents derived from natural sources such as lignocellulosic hydrolysates, wheat/rice straw extracts, etc. The natural media may be used or added externally into the artificial media as well.

The present invention in its one aspect provides a novel non-clostridial bacteria belonging to Providencia species having Accession No. MCC0049. In another aspect the present invention provides for a process of industrial scale production of biofuels from biomass using novel non-clostridial bacteria belonging to Providencia species MCC0049. The biomass according to the invention is lignocellulosic biomass. In addition, the present invention discloses a process for isolating the non-clostridial bacterial strain from soil. In another aspect the present invention provides for a process of acclimatizing a novel strain of non-clostridial strain of bacteria belonging to Providencia species MCC0049.

Thus another aspect of the present invention provides for a non-clostridial strain of bacteria belonging to Providencia species deposited at Microbial Culture Collection at National Centre for Cell Science designated as Accession No.MCC0049 (herein also designated as Providencia strain MCC0049) dated 2-January-2015.

Further, in another aspect the present invention also provides Providencia strain MCC0049 having following characteristics:
(a) Size: Rod-shaped, approximately 1.5-2.5 µm in size (Figures 8a-c).
(b) Gram Staining: Gram negative, non-spore forming.
(c) Growth conditions: The bacterial can grow both aerobically and anaerobically in suitable media such as pure carbohydrates e.g. glucose and xylose etc (Figure 6a and b).
(d) Sequencing: Upon 16srRNA sequencing the strain Providencia strain MCC0049 showed 99.6% identity with Providencia sp.

Another aspect of the present invention provides for production of biofuel under both aerobic and anaerobic conditions using the non-clostridial bacterial strain as herein described. In another aspect the present invention provides for novel strain as herein described which is highly resistant to biofuel and therefore is able to produce higher concentrations of the biofuel. Accordingly, the unique advantage of the novel strain as herein described in the present invention lies with its ability to withstand and even further grow in the presence of the biofuel produced by it.

In other words the bacterial strain as herein described utilizes the raw material for growing the said strain and convert it into biofuel, yet the said bacterial strain does not loose capability of further growth in the presence of biofuel and thus can be recovered efficiently for further use. This in another aspect the present invention provides a novel strain as herein described which is highly resistant to biofuel, in particular to n-butanol and therefore produces higher concentrations of the biofuel up to its tolerance levels (Figure 5).

In another aspect the present invention provides a novel bacterial strain as herein described for the production of biofuel, wherein the strain is capable of fermenting in a wide range of media including synthetic or natural media. Natural media includes lignocellulosic hydrolysate/biomass such as but not limited to rice, wheat straw, sugarcane bagasse and sorghum.

In another aspect the present invention provides a novel bacterial strain for the production of biofuel, wherein the strain is capable of fermenting a wide range of carbohydrates present in synthetic or natural media. The sugars in natural media i.e. lignocellulosic hydrolysate/biomass of but not limited to rice, wheat straw, sugarcane bagasse and sorghum could be glucose, xylose, cellobiose, arabinose, cellulose.

In another aspect the present invention provides a novel non-clostridial bacterial strain as herein described, wherein the bacterial strain can produce biofuel particularly butanol and ethanol under both aerobic and anaerobic conditions in greater concentrations and at higher rates. Further the bacterial strain is resistant to high concentration of butanol.

The present invention provides a novel Providencia strain having Accession No. MCC0049 for the production of biofuels, in particular butanol under both aerobic and anaerobic conditions. According to the present invention the isolated strain produces butanol in the range of 2-8 g/L as shown in Figure 1d.

Further the strain of the present invention as herein described produces butanol and ethanol under aerobic and anaerobic condition, with limited formation of by products such as acetone (Figures 4a-c). In another aspect the present invention provides a novel strain of Providencia having Accession No. MCC0049 which has high biofuel, particularly butanol, tolerance up to 3% (Figure 5). In accordance with the invention, the novel strain as herein described is able to tolerate the biofuel concentration from about 0.0001 to about 3% (Figure 5).

In another aspect the present invention provides for novel strain of Providencia having Accession No. MCC0049 which isolated and then acclimatized to grow under in vitro conditions. The method of acclimatization involves collecting samples of soil from Paddy fields followed by heat treating the samples to inhibit vegetative cells inoculating the treated samples, incubating the treated samples overnight, plating them on TSB agar plates under aerobic conditions and reinforced clostridial medium under anaerobic conditions, incubating to obtain pure colonies, picking and inoculating the individual colonies into medium, supplementing with glucose and urea and incubating under both aerobic and anaerobic conditions, and selecting the microbial strain producing butanol under both, aerobic and anaerobic conditions.

Another aspect of the present invention provides for production of butanol from the novel Providencia strain MCC0049.
Another aspect of the present invention provides for the process of producing butanol/ethanol using isolated strain of Providencia sp MCC0049 which involves repeated sub-culturing of said strain in a medium, fermenting the purified strain in suitable medium such as hydrolysate under optimum conditions of temperature and pH to obtain butanol both aerobically and anaerobically, collecting the supernatant from aerobic and anaerobic cultures every 24 hours, and analysing for biofuel preferably butanol production by Ultra High Performance Liquid Chromatography (UHPLC) attached to Refractive Index Detector (RID).

According to the invention, the strain Providencia sp MCC0049 produces butanol in the range of 2-8 g/L (Figure 1d). Figure 1a and 1c shows the amount of butanol produced aerobically and anaerobically respectively.

The hydrolysate used as media for biofuel production is produced by thermochemical pretreatment of selected biomass using acids such as sulphuric or phosphoric or dilute ammonia in the range of 0.5-2 % w/v, heated at 120oC-150oC for a period of 15 min-60 min.

Further disclosed herein, is a method for analysis of butanol produced by Providencia sp MCC0049 using High Performance Liquid Chromatography. Collected samples from batch fermentation cultures at different time intervals were centrifuged at 20,000g for 15 minutes at 4oC, around 200µl of supernatant is loaded in eppendorf 96 well plates and 5µl of sample was injected for solvent analysis in Agilent 1290 Infinity series Ultra High Performance Liquid Chromatography equipped with Quaternary pump, Auto sampler maintained at 4oC, and analysed using Agilent Zorbax Stable Bond- Aq Rapidi Resolution High Definition columns, maintained at 35oC-40oC, with completely aqueous mobile phase having 0.03mM sulphuric acid (H2SO4) and 0.001% formic acid and Refraction Index Detector maintained at 40oC-45oC (Always 5oC more than column temperature).

Accordingly, the main embodiment of the present invention provides for a novel non-clostridia bacterial strain of Providencia species having Accession No. MCC0049 that can grow under both aerobic and anaerobic conditions.

Another embodiment of the present invention provides for a novel strain as herein described for producing biofuels.

Another embodiment of the present invention relates to a novel strain as herein described for producing biofuels under both aerobic and anaerobic conditions.

Yet another embodiment of the present invention provides for a process of producing biofuel, said process comprising the steps of:
(a) growing the strain of Providencia species MCC0049 in a culture medium under aerobic or anaerobic conditions at a temperature of 30-35°C at a pH of 5-8 for about 12-24 hours; and
(b) obtaining biofuel.

Yet another embodiment of the present invention provides for a process as herein described, wherein for the aerobic and anaerobic conditions culture medium is selected from T6 medium, LB medium, TSB Medium or Reinforced Clostridial Medium.

Yet another embodiment of the present invention provides for a process as herein described, wherein the biofuel obtained is butanol, ethanol and acetone or mixture thereof.

Yet another embodiment of the present invention provides for a process as herein described, wherein the biofuel obtained is in the range of 2-4 g/L.

Yet another embodiment of the present invention provides for a process as herein described, wherein the biofuel is butanol having a concentration range of 2-4 g/L.

Yet another embodiment of the present invention provides for a process as herein described, wherein the step (a) the pH is 7.

Yet another embodiment of the present invention provides for a process as herein described, wherein the step (a) the temperature is 30-32°C.

Another embodiment of the present invention provides for a novel strain Providencia species MCC0049 for production of butanol biofuel.

Another embodiment of the present invention provides for a method acclimatization of novel strain of Providencia species strain MCC0049, said method comprising the steps of:
(a) isolating the Providencia species strain MCC0049 from the soil samples;
(b) growing the Providencia species strain MCC0049 in an aerobic or anaerobic culture medium at the temperature of 25-37°C at a pH of 5-8; and
(c) obtaining acclimatized pure colonies of Providencia species MCC0049.

Another embodiment of the present invention provides for a method of acclimatization as herein described, wherein the aerobic and anaerobic culture medium is selected from LB medium, TSB medium, Thioglycoate medium, T6 medium supplemented with 1-2% urea and 1-6% glucose, or reinforced clostridial medium.

Another embodiment of the present invention provides for a method of acclimatization Providencia species strain MCC0049, wherein the acclimatized Providencia species strain MCC0049 is useful for producing biofuels.

Another embodiment of the present invention provides for a method of acclimatization of Providencia species strain MCC0049, wherein the acclimatized Providencia species strain MCC0049 is useful for producing butanol.

While this invention has been described in detail with reference to certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments rather, in view of the present disclosure, which describes the current best mode for practicing the invention, many modifications and variations, would present themselves to those skilled in the art without departing from the scope and spirit of this invention.

In the following section preferred embodiments are described by way of examples to illustrate the process of the invention. However, these are not intended in any way to limit the scope of the present invention.

Example 1
Acclimatization of isolation of strain of Providencia sp MCC0049.
Soil samples are collected from the Paddy fields of Kadapa region of India, and heat treated at around 75-85oC to inhibit vegetative cells. The heat treated soil samples are then inoculated in Luria Broth and incubated overnight at 25oC-37oC. 10µL of the culture is then plated on Trypticate soya agar plates under aerobic conditions, Luria Bertani medium, anerobic thioglycolate medium, and reinforced clostridial medium under anaerobic conditions and incubated at 30oC-35oC till pure acclimatized colonies are obtained.

Example 2
Production of Butanol
For anaerobic conditions, the culture stocks prepared under anaerobic conditions are handled in the anaerobic chamber (BacBASIC). Individual colonies are picked and inoculated into T6 medium (Composition per Litre: tryptone-6g, yeast extract-2g, potassium phosphate-0.5g, magnesium sulphate-0.3g, ferrous sulphate-10mg, ammonium acetate-3g, cysteine hydrochloride-0.5g.) supplemented with 1-6% Glucose and 1%-2 % urea or Luria Bertani medium supplemented with 1-6% glucose and 1-2% urea or Reinforced clostridial medium supplemented with 1-5% glucose and 1-2% urea, TSB supplemented with 1-5% glucose and 1-2% urea and incubated between 30°C-35oC, preferably around 32oC and pH around 5-8, preferably around 6 under nitrogen atmosphere to create anaerobic conditions. The supernatant is collected from anaerobic cultures every 12 hours upto 60 hours and analysed for butanol production by UHPLC attached to RID. The Figure 1a provides butanol production under anaerobic conditions by the Providencia sp MCC0049 strain. Further strain purification is done by repeated sub-culturing of Providencia sp MCC0049 in LB medium.

Example 3
Strains producing butanol under aerobic conditions were inoculated into T6 medium (Composition per Litre: tryptone-6g, yeast extract-2g, potassium phosphate-0.5g, magnesium sulphate-0.3g, ferrous sulphate-10mg, ammonium acetate-3g, cysteine hydrochloride-0.5g.) supplemented with 3-5% Glucose and 1% -10% urea or Luria Bertani medium supplemented with 1-5%glucose and 1-10% urea or Reinforced clostridial medium supplemented with 1-5% glucose and 1-10% urea, TSB supplemented with 1-5% glucose and 1-10% urea and incubated at 32oC -35oC, preferably at 32oC at 180-220 RPM, preferably at 180 rpm and under 0.5 vvm air to create aerobic conditions. The supernatant is collected from aerobic cultures every 12 hours and analysed for butanol production by UHPLC attached to RID (Figure 1a). The Figure 1c provides butanol production under aerobic conditions by the Providencia sp MCC0049 strain.

Example 4
Analysis of Butanol
Solvents produced by fermentation are quantified using Agilent Ultra High Performance Liquid Chromatography. 1 ml samples are collected at every 24 at every 6 hours and 12 hour intervals and centrifuged for 15-30 minutes at 14,000 rpm (20817 g). 100-200µl of the supernatant is aliquoted in a 96 well plate and analysed for solvent production (samples in 96 well plate maintained at 4oC). Only 5 µl of the supernatant is used for solvent analysis by UHPLC.
A standard curve was plotted by preparing aqueous mixture of acetone, butanol and ethanol in various concentrations. The butanol present in the supernatant was analysed by comparing to the standard curve (Figures 7a-e).

Example 5:

A comparative study of the biofuel production between the novel strain Providencia sp MCC0049 strain and conventional strain shows that Providencia sp MCC0049 strain is high efficacy for the production of butanol in comparison to the convention strain as shown in Table 1 below and Figures 2-3:
Table1: Comparison of butanol production of Providencia sp MCC0049 and conventional butanol producer Clostridium acetobutylicum NCCS2337.
Strain Amount of butanol produced in grams/L Condition
Providencia sp MCC0049 5.2g/L Aerobic and microaerophillic conditions
Clostridium acetobutylicum NCCS2337 2.27g/L Strictly anaerobic