Production of Biofuel from sugar tolerating Honeybee gut bacteria.

Production of biofuels and chemicals through a fermentation-based manufacturing process requires renewable feedstock such as corn stover, sugarcane bagasse and plant biomass lignocellulose to generate biofuel. But this process requires the availability of microorganisms that can convert the car

Project Title

Production of Biofuel from sugar tolerating Honeybee gut bacteria.

Project Area of Specialization

Biomedical Engineering

Project Summary

Production of biofuels and chemicals through a fermentation-based manufacturing process requires renewable feedstock such as corn stover, sugarcane bagasse and plant biomass lignocellulose to generate biofuel. But this process requires the availability of microorganisms that can convert the carbon source into desired fuel. The microorganism that can survive in a sugar-rich environment can be contemplated as the best choice for the production of biofuel. Previously, yeast Saccharomyces cerevisiae and Zymomonas mobilis are common to produce bioethanol but these days bacterial strains are best suited for the production of biofuels because of their ability to naturally utilize a variety of carbon sources (including sugars and sugar alcohols) under both aerobic and anaerobic conditions and

This study will use the bacterial flora of the honeybee gut for biofuel production. There are about 300 to 1000 distinct types of bacteria are found in the honeybee gut. Gram-negative Proteobacteria (e.g. Snodgrassella and Gilliamella ssp.) and Gram-positive Firmicutes are the most prevalent bacteria discovered in (queen) adult honey bees.

These bacteria have a role in the breakdown of complex macromolecules such as polysaccharides and polypeptides into sugar and amino acids respectively. These bacteria are considered Sucretolerant microbes that grow in the presence of high sugar concentrations and lower water activities.

 The aim of this study is to identify the sugar tolerating bacteria in the honeybee gut. These bacterial strains with high efficiency of sugar utilization and tolerance can be used to produce a variety of biofuels and chemicals. The future outcomes of this study can also be tuned to different types of biomass production by laboratory adaptive evolution.

Project Objectives

The aim of study is to identify the industrially important sugar tolerating bacteria from honeybee gut. The theme of present research is:

1. To isolate bacteria from honeybee gut.
2. To grow them in the presence of high sugar media.
3. To separate them from non-sugar fermenting bacteria.
4. To identify them by 16s ribosomal RNA sequencing.
5. To analysis their potential of biofuel production by HPLC UV detector at ?max 230?nm.

Project Implementation Method

METHODOLOGY

1. Sample collection and dissection of the bee.

Honeybees will be dissected, and the dissected guts were macerated with sterile dissection scissors in 0.8% NaCl solution and immediately stored at -20 °C.

2. Culturing of bacteria in Luria Bertani media.

From the preserved bee gut samples dilution will be made. Diluted samples were inoculated in LB (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 15 g agar, and 1 liter of distilled water) agar plates and incubated for 24–48 h at 37 °C. The separated colonies in master plates will be sub-cultured in LB agar plates and incubated at 37 °C.

3. Biochemical tests.

Various biochemical tests will be performed including the Coagulase test, Oxidase test, Urease test, Lactose fermentation test, hydrogen peroxide production test etc. for the identification of bacterial isolates for non-fastidious, non-enteric Gram-negative rods.

4. Preparation of sugar-rich media.

Solutions of fructose, sucrose, and glucose with concentrations of 15%, 30%, and 50% will be prepared and mixed in LB media respectively in specific volumes. The sugar fermenting bacteria will be grown in this media for the separation of sugar tolerating bacteria from non-sugar tolerating bacteria.

5. Identification of sugar tolerating bacterial species by 16s rRNA sequencing.

For the identification of sugar tolerating bacterial species, the isolated colony will be subjected to PCR amplification of the 16S rRNA gene using forward primer 5’-GGCTCAGAACGAACGCTGGCGGC-3’ and the reverse primer 5’-C CCACTGCTGCCTCCCGTAGGAGT-3’.The sequencing data will be analyzed by the BLAST tool for specie identification

6. Analysis of biofuel production by HPLC.

Bacterial culture in sugar-rich LB media will be centrifuge and the supernatant will be used to determine the sugar derivative using the C18 column and a binary solvent system comprising 0.5% aqueous trifluoroacetic acid as solvent A and acetonitrile as solvent B in the gradient mode in HPLC UV detector at ?max 230?nm. These cultures will be tested production of biofuel using agricultural residues.

Benefits of the Project

One of the major challenges in the modern world is the unsustainable dependence on petroleum which serve as the primary source of fuel for the transportation of and synthesis of many other chemicals like solvents, fertilizers, pesticide, and plastics. To fulfill the future demand of the society we required a sustainable supply of energy. Through the fermentation process, microbes can be used to generate biofuel by fermentation of sugars.

Honeybee gut bacteria are highly osmotolerant. They can survive in a high sugar environment of honey and metabolize and ferments these sugars by breaking them into simpler products. These gut microbes can be used to degrade nonfood agricultural residues like corn stover, sugarcane bagasse, and plant biomass lignocellulose to generate biofuel from glycolysis or pentose phosphate pathway. The conversion of these agricultural residues into fuels by sugar tolerating bacteria of the honeybee gut is a cost-effective way to produce biofuel.

Technical Details of Final Deliverable

• The purchase of chemicals and instruments will expect to be finished before the start project.
• sample collection and bacterial isolation.
• Identification of sugar tolerant bacteria by DNA sequencing.
• Production of sugar derivatives and biofuel
• manuscript writeup and submission

Final Deliverable of the Project

Software System

Core Industry

Petroleum

Other Industries

Core Technology

Others

Other Technologies

Sustainable Development Goals

Affordable and Clean Energy, Industry, Innovation and Infrastructure

Required Resources

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