Microbial Induced Biotechnological Processes for Biofuel Production from Waste Organics Conversion

In the current era there are huge quantities of waste organic matter available, creating a big burden to the environment. To address these issues, researchers started to apply effective and microbial induced biotechnological processes that can mitigate these waste matters. In this context, different nature of microbial systems are involved in hydrolysing the waste organic material into fermentable sugar. These can be easily consumed by specific microbial systems like Saccharomyces cerevisiae MTCC 3821 and Clostridium acetobutylicum that produced bioethanol and biobutanol, respectively. Saccharomyces cerevisiae was cultured in specific media and incubated at rotary shaker with 150 rpm at 30°C for 72 to 96 hours. Ethanol concentrations from different waste matters were found in the range of 1.2-1.5 g.L -1 . Ethanol synthesis was done by shake flask experiment with addition of glucose (50 g.L -1 ) to waste organic hydrolyzed solution. Non-glucose media produced less than 3 g.L -1 ethanol but glucose media produced 4.5 g.L -1 . Next , Clostridium acetobutylicum was grown in culture media containing waste organics as sole carbon substrate with pH 7 and then was incubated in anaerobic conditions at 35°C for 72 hours, produced butanol (0.7 to 1.25 g.L -1 ). This research work promoted biofuels synthesis by keeping a waste mitigation strategy.


INTRODUCTION
The global energy demand has increased to 1.3% in 2022 and more than 82% of this energy supply demand is fulfilled by fossil fuels sources.The major global energy requirement can be fulfilled by non-renewable energy sources like crude oil, coal and natural gas.However, these resources are non-renewable, take millions of years for formation and could be exhausted in near future. 1 Rising price of crude oil, various environmental challenges and political instability are the other factors that forced the replacement of fossil fuel with other eco-friendly alternative energy sources.Since the dawn of time, biofuels have been used as an energy source.Biofuels marked their presence from the last few decades with firing and heating energy source, being discovered.During the early 20 th century, Henry Ford proposed automobiles, using bioethanol as their fuel source for future needs. 2 In 1906, the New York Times published a description of ethanol as the fuel source for automobiles and published a report in ″Auto Club Aroused Over Alcohol Bill″.During World War I and II, decrease in raw materials and natural resources forced the production of ethanol as an alternative to fossil product derived petrol.The production of bio-ethanol was started and now increased to 600 million gallons with sequential improvement in yield/ titer. 2,3Biofuels can be produced from any photosynthetic living organisms like photosynthetic bacteria (e.g., Cyanobacteria, Proteobacteria), algae and vascular land plants.Biofuels produced can be solid, liquid and gaseous nature's fuels. 4iquid biofuels include ethanol, methanol, biodiesel and gaseous fuels (include biohydrogen and methane).Liquid biofuels are used as fuel for vehicles, fuel cells and also in fuel engines.Biofuels are classified as primary secondary biofuels and others. 5The energy derived from plant products like food grains and fruits juices can be reported as primary biofuel.Secondary biofuels are produced indirectly derived from plant waste matters. 4,5iofuels are further classified based on origin and technology aspects as first, second, third and fourth generation biofuel.In 2022, global production of biofuels was increased by 46% (i.e., 1.9 million barrels of oil equivalent per day. 6his research has discussed the biofuel synthesis from waste biomass conversion and microbial fermentation.

Biofuels and its synthesizing parameters
Biofuel is the fuel, extracted from food grains or waste biomass that comes from plant or animal sources.It can be found as a solid, liquid or gaseous nature.Based on the source of waste biomass/organic matter, biofuels originate with their production as primary and secondary biofuels or other types.Primary biofuels can be biosynthesized from the edible plant foods/ products and its waste biomass.But other forms of biofuel can be synthesized from animal waste, wood or crop waste and these can be used as raw carbon sources with high potential substrates for energy production (heat or electricity production). 7iofuels are further classified as first, second, third and fourth generation biofuel, on the basis of feedstock used.Produced biomasses can come from living organisms especially from plants, and microorganisms, which harness solar energy and convert into chemical form and stored organic matter in their body.This energy is harnessed and converted into electrical or heat energy.Bioethanol and biodiesel are the most studied biofuels and also utilized in transportation tasks. 8irst generation bioethanol (C 2 H 5 OH/ EtOH) is produced from the fermentation process of wheat grain and sugarcane juices.This generation of bioethanol is primarily used as transportation fuel in its pure form or blended with gasoline (e.g., gasoline blended with 10% ethanol, as E10).Replacing petroleum products with this generation of bioethanol can reduce the emission of carbon dioxide in two ways like heat, steam and carbon dioxide production after combustion of bioethanol. 7,8And the carbon dioxide present in the atmosphere is utilized for growing crops as raw material.Therefore, bioethanol is considered to be a carbon neutral fuel source.In 2022, at worldwide level bioethanol production has reached 28 billion gallons, with the trend of an increasing level (850 billion) from previous year and it is the highest in the last seven years. 9First generation bioethanol can be produced either by sugar or its products containing crops such as palm juice, sugarcane or starch containing crops such as wheat, barley, rice.As starch can't be directly converted into ethanol due to long chain polymer structure.The starch biomass is degraded by using two enzymes.Starch is converted into dextrin and oligosaccharides with the help of amylases.Dextran is converted into glucose by enzymes like glucoamylase and yeast can be added for fermentation process. 10econd generation biofuels can be synthesized by hydrolysing plant waste biomass and it needs an effective pretreatment and microbial fermentation system.In this fuel bioproduction, yeast and bacterial strains are utilized for fermenting the glucose to ethanol or other alcoholic fuels.And environment reports for high availability waste organics are reports.For utilization of algal biomass for third generation biofuel is necessary. 9,10The algae are cultivated in open shallow raceway ponds attached with wheels for better aeration and nutrient circulation.These ponds are easy to operate and require less operating cost.Unfavorable photosynthetic microbes can enter the pond through atmosphere or waterways and result in low yield of algae. 9,11able 1 discusses some algal species that have contributed to biodiesel and Table 2 discusses the different generation biofuels that used in transport engine fuels.

MATERIALS AND METHODS
For this research work, first Saccharomyces cerevisiae MTCC 3821 was incubated in YEPD (yeast extract peptone dextrose) media at 30°C.YEPD media consisted of yeast extract powder (10 g.L -1 ), peptone (C 13 H 24 O 4 ~20 g.L -1 ), D-glucose (dextrose ~20 g.L -1 ), chloramphenicol (500 mg/L) with then keeps media pH of 5.5.It was adjusted with 1.0 N H 2 SO 4 / HCL solution.For YEPD agar plate development, agar-agar (20 g.L -1 ) was added.The sub-culturing was done every 15 days.For culture preparation, three loopful of Saccharomyces cerevisiae MTCC 3821, strain into growth medium (100 ml) is done and incubated at 30°C, 200 rpm and 24 hours to obtain exponential phase.Butanol production task was done by adding Clostridium acetobutylicum strain and this strain was isolated from local agricultural land of Vishakhapatnam by using particular media.Single strain is isolated and cultured in CGM (clostridium growth media) under anaerobic conditions.The pure culture was done for gram-staining tasks and microscopic characteristics observation.Rod-shaped and gram-positive strains were selected for further study.For this microbial strain growth, P2 medium was used.The sub-culturing was done every 10 days.Solvent production capabilities were identified by using acetone tests.Positive acetone production was identified by yellow to purple colour change by adding 1 ml of 5% of sodium nitroprusside (SNP ~Na 2 [Fe(CN) 5 (NO)]) and a drop of 40% NH 4 + solution.Biobutanol production has occurred in P2 medium.All 15 strains are cultured into 10 ml of P2 medium broth and incubated under anaerobic condition for 7 days at 37°C.Butanol concentration was checked by HPLC techniques.

Fermentation/biotechnological process conditions for biofuel synthesis
Saccharomyces cerevisiae MTCC 3821 were inoculated in fermentation broth and incubated in a rotary shaker with 200 rpm speed rate under partial anaerobic conditions at 30°C for 48 hours.In every 6 hours, the sample analysis was performed to analyse for ethanol/residual sugars.During the complex organic matter hydrolysis, the effect of inhibitory compounds on ethanol yield was also compared.Biomass concentration was determined by a UV spectrophotometer at 600 nm.Supernatants were obtained by centrifugation of the sample at 5000 rpm for 15 min.Supernatants are filtered by using a 0.22 µm membrane filter.Ethanol and residual sugar concentrations were determined by HPLC technique with Rezex ROA organic acid, 300 × 7.8 mm HPX-87H column, and RI detector.Among many strains of Clostridium acetobutylicum, only one was isolated and it showed highest butanol concentration in ABE production.They were cultured in P2 medium with waste organic hydrolysate as the sole carbon source and incubated in anaerobic conditions at 37°C for 72 hours.To study the effect of inhibitory compounds like formic acid, the fermentation of detoxified pretreated hydrolysate and nonremoval was compared.Samples were collected, centrifuged at 6000 rpm for 5 min and supernatant was collected for analysis of acetone, butanol, ethanol and reducing sugar by HPLC.

Ethanol production
During experiments, when Saccharomyces cerevisiae MTCC 3821 was inoculated in untreated waste organic hydrolysate, low ethanol yield was obtained.It may be due to the presence of some inhibitory compounds in the hydrolysate media.Different concentration of ethanol was compared for sugarcane bagasse, rice straw and wheat straw hydrolysate solution and the highest ethanol yield was reported by rice straw at pH 5.5.The rice straw at pH 5.5 has produced the ethanol of 2.25 g.L -1 while the sugarcane bagasse based hydrolysate ethanol yield was found to 2.12 g.L -1 which is comparatively low.The wheat straw at the same pH has produced 1.98 g.L -1 of ethanol.In general, S. cerevisiae is an acidophilic organism and it requires an acidic medium for its growth and development.The optimum pH for S. cerevisiae growth was 4.0-6.0,depending on the temperature, oxygen availability and the type of strain.Optimum pH is important for all cellular activities especially for enzymes and transport proteins bound to plasma membranes.During growth and metabolism of S. cerevisiae, this microbe needs to maintain a constant pH during fermentation period.The environmental pH (pH of media) changes needs to maintain the H + ion concentration by diffusion.When outside pH can be varied too much from the optimum range, then the cell cannot maintain the intracellular pH, affecting the functioning of enzymes.Furthermore, it results in denaturation of enzymes and cells  S. cerevisiae requires a temperature range of 27°C-40°C for the fermentation process that results in the best yield and titer.Maximum ethanol yield (2.24 g.L -1 ) was obtained from plant origin waste organic matter at 72 hours.Fermentation of rice and wheat straw hydrolysis solution has produced ethanol of different titers (1.85 g.L -1 and 1.0 g.L -1 respectively).Figure 1 shows ethanol production from waste matter in crops.
Biobutanol synthesis H i g h co n c e nt rat i o n o f b u ta n o l (1.2 g.L -1 ) and ABE (1.9 g.L -1 ) in 50 ml of biomass hydrolysate solution was achieved in production /fermentation media that was detoxified before start to fermentation.A low concentration of butanol (0.35 g.L -1 ) and ABE (0.8 g.L -1 ) in 10 ml of biomass hydrolysate solution was found in nondetoxified solution also.But, some improvement in concentration of butanol (1.4 g.L -1 ) and ABE (2.1 g.L -1 ) in 50 ml of biomass hydrolysate solution in production/fermentation media that was systematically detoxified.Figure 2 shows the titers of butanol from waste matter in crops.From these experiment results, it has indicated low concentration of fermentation production was due to low initial concentration of biomass hydrolysates and it was attributed low amount of carbon or glucose sources as impact of mass action.Clostridium species has shown the capability of clostridial solvent production and it occurs in a biphasic process.In this process, the first phase (i.e., acidogenic phase) is reported to acid forming pathways with full activation and it is responsible for production of acetate, butyrate, hydrogen and carbon dioxide as major products.In the first phase, the clostridium growth period is found during the exponential growth phase.And then the second phase is started as a solventogenic phase and this phase is characterized by assimilation of acids and then this process is responsible for production of acetone, butanol and ethanol (ABE).

CONCLUSION
This research work was done on a laboratory scale and authors have done some sets of experiments on biofuel production, such as ethanol.During this work, systematic pretreatments and enzymatic hydrolysis tasks were performed in the laboratory.This work focussed on the mitigation of waste organic matter in the environment that was generated during crop production and food processing.Saccharomyces cerevisiae MTCC 3821 was inoculated in a different nature of waste organic hydrolysate and low ethanol yield was obtained in non-detoxified biomass hydrolysates.Maximum ethanol yield (2.24 g.L -1 ) was obtained from plant origin waste organic matter at 72 hours in detoxified broth.Ethanol of different titers in fermentation of rice (1.85 g.L -1 ) and wheat straw (1.0 g.L -1 ) hydrolysed solution were produced.

Figure 1 .
Figure 1.Ethanol production from synthetic media (A) and biomass hydrolysate containing media (B) sugarcane waste and (C) rice waste

Table 1 .
Different microalgal biomass utilization for third generation biofuels production

Table 2 .
Different biofuels from different waste organic sources and its impacts